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- EMDB-5559: Cryo-em map of one molecule of factor VIII light chain from helic... -

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Basic information

Entry
Database: EMDB / ID: EMD-5559
TitleCryo-em map of one molecule of factor VIII light chain from helically organized factor VIII light chain molecules bound to lipid nanotubes
Map dataCropped 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
  • Sample: Cropped volume corresponding to 2x1 factor VIII light chain molecules from EMD-5540
  • Protein or peptide: blood coagulation Factor VIII light chain
Keywordsmembrane 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-coated ER to Golgi transport vesicle / COPII-mediated vesicle transport / 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 / endoplasmic reticulum lumen / copper ion binding / extracellular space / extracellular region / plasma membrane
Similarity search - Function
Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Multicopper oxidase, C-terminal / Multicopper oxidase ...Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Multicopper oxidase, C-terminal / Multicopper oxidase / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Multicopper oxidase, N-terminal / Multicopper oxidase / Cupredoxin / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Coagulation factor VIII
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsStoilova-McPhie S / Lynch GC / Ludtke S / Pettitt BM
CitationJournal: 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
DepositionDec 21, 2012-
Header (metadata) releaseMay 15, 2013-
Map releaseAug 28, 2013-
UpdateAug 28, 2013-
Current statusAug 28, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j2s
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5559_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5559.map.gz / Format: CCP4 / Size: 330.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCropped 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

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.9 Å/pix.
x 28 pix.
= 81.2 Å		2.9 Å/pix.
x 54 pix.
= 156.6 Å		2.9 Å/pix.
x 57 pix.
= 165.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.001 / Movie #1: 0.001
Minimum - Maximum-0.01030504 - 0.01894764
Average (Standard dev.)0.00152538 (±0.00413782)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-39-29-21
Dimensions545728
Spacing545728
CellA: 165.3 Å / B: 156.6 Å / C: 81.200005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z575428
origin x/y/z0.0000.0000.000
length x/y/z165.300156.60081.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-29-39-21
NC/NR/NS575428
D min/max/mean-0.0100.0190.002

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Supplemental data

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Sample components

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Entire : Cropped volume corresponding to 2x1 factor VIII light chain molec...

EntireName: Cropped volume corresponding to 2x1 factor VIII light chain molecules from EMD-5540
Components
  • Sample: Cropped volume corresponding to 2x1 factor VIII light chain molecules from EMD-5540
  • Protein or peptide: blood coagulation Factor VIII light chain

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Supramolecule #1000: Cropped volume corresponding to 2x1 factor VIII light chain molec...

SupramoleculeName: 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
Molecular weightExperimental: 80 KDa / Theoretical: 80 KDa / Method: SDS-PAGE

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Macromolecule #1: blood coagulation Factor VIII light chain

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: blood plasma
Molecular weightExperimental: 80 KDa / Theoretical: 80 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: CHO
SequenceUniProtKB: Coagulation factor VIII

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: 20 mM Tris-HCl, 150 mM NaCl, 5mM CaCl2
GridDetails: 300 mesh R2x2 Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK III / Method: Blot 3.5 seconds before plunging
DetailsThe protein was mixed in 1:1 w/w ratio with lipid nanotubes solution

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureMin: 93 K / Max: 103 K / Average: 99 K
Alignment procedureLegacy - Astigmatism: corrected at 400,000 times magnification
DateApr 2, 2010
Image recordingCategory: 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 beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsIHRSR, SPIDER and EMAN2
Final reconstructionApplied 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 correctionDetails: particle stacks for each micrograph were corrected for CTF (only phase correction)

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Atomic model buiding 1

Initial modelPDB ID:

3cdz


Chain - Chain ID: B
SoftwareName: UCSF-Chimera, VMD
DetailsThe 3CDZ chain B coordinates were fitted flexibly within the 3D map with the 'fit to volume' option of the UCSF Chimera software.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: optimal fit
Output model

PDB-3j2s:
Membrane-bound factor VIII light chain

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