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

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Basic information

Entry
Database: PDB / ID: 3j2s
TitleMembrane-bound factor VIII light chain
ComponentsCoagulation factor VIII light chain
KeywordsBLOOD CLOTTING / blood coagulation / cofactor / factor VIII / hemophilia / membrane binding
Function / homology
Function and homology information


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 / 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 / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 sulfation at Y1699 / 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
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-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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 15 Å
AuthorsStoilova-Mcphie, S. / Lynch, G.C. / Ludtke, S. / Pettitt, B.M.
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, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2013Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id

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Structure visualization

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Assembly

Deposited unit
B: Coagulation factor VIII light chain


Theoretical massNumber of molelcules
Total (without water)74,0351
Polymers74,0351
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Coagulation factor VIII light chain / Antihemophilic factor / AHF / Procoagulant component / Factor VIIIa light chain


Mass: 74035.359 Da / Num. of mol.: 1 / Fragment: UNP residues 1710-2356
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F8, F8C / Cell (production host): ovary / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00451
Sequence detailsTHE AUTHORS STATE THAT F1899L IS A NATURAL VARIANT.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1Factor VIII light chain helically assembled onto lipid nanotubesCOMPLEX0
2blood coagulation Factor VIII light chain1
Buffer solutionName: 20 mM Tris-HCl, 150 mM NaCl, 5mM CaCl2 / pH: 7.4 / Details: 20 mM Tris-HCl, 150 mM NaCl, 5mM CaCl2
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 300 mesh R2x2 Quantifoil grids
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 95 K / Humidity: 100 %
Details: Blot for 3.5 seconds before plunging into liquid ethane (Vitrobot Mark III)
Method: Blot for 3.5 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: JEOL 2010F / Date: Apr 2, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 52000 X / Calibrated magnification: 52000 X / Nominal defocus max: -4400 nm / Nominal defocus min: -700 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: GATAN LIQUID NITROGEN / Temperature: 95 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 16 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Details: 4096 x 4096 pixels @ 15 microns per pixel
Image scansNum. digital images: 69

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2EMAN23D reconstruction
3IHRSR3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: phase corrected based on first Thon ring
Helical symmertyAngular rotation/subunit: 10 ° / Axial rise/subunit: 8 Å / Axial symmetry: C1
3D reconstructionResolution: 15 Å / Nominal pixel size: 2.9 Å / Actual pixel size: 2.9 Å / Magnification calibration: TMV images / Details: IHRSR combined with SPIDER and EMAN2 / Symmetry type: HELICAL
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms5125 0 0 0 5125

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