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- EMDB-3027: Electron cryo-microscopy of porcine Factor VIII bound to lipid na... -

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Basic information

Entry
Database: EMDB / ID: EMD-3027
TitleElectron cryo-microscopy of porcine Factor VIII bound to lipid nanotubes and helical reconstruction
Map dataHelical reconstruction of porcine Factor VIII bound to LNT as described in the submitted references.
Sample
  • Sample: Helically organized porcine blood coagulation Factor VIII bound to a lipid nanotube
  • Protein or peptide: Blood coagulation Factor VIII
  • Ligand: lipid nanotubes
KeywordsBlood coagulation factor VIII / Lipid nanotubes / Cryo-electron microscopy / Membrane-bound organization / Helical reconstruction
Biological speciesSus scrofa (pig) / unidentified (others)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 15.5 Å
AuthorsDalm D / Galaz-Montoya JG / Miller JL / Grushin K / Villalobos A / Koyfman AY / Schmid MF / Stoilova-McPhie S
CitationJournal: J Vis Exp / Year: 2014
Title: Helical organization of blood coagulation factor VIII on lipid nanotubes.
Authors: Jaimy Miller / Daniela Dalm / Alexey Y Koyfman / Kirill Grushin / Svetla Stoilova-McPhie /
Abstract: Cryo-electron microscopy (Cryo-EM)(1) is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment(2). Coagulation Factor VIII ...Cryo-electron microscopy (Cryo-EM)(1) is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment(2). Coagulation Factor VIII (FVIII)(3) is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting(4). Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete(5). Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa(6,7). In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.
History
DepositionMay 27, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseAug 12, 2015-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image3027.png

Downloads & links

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Map

FileDownload / File: emd_3027.map.gz / Format: CCP4 / Size: 41.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of porcine Factor VIII bound to LNT as described in the submitted references.
Voxel sizeX=Y=Z: 2.9 Å
Density
Contour LevelBy AUTHOR: 0.0036 / Movie #1: 0.0036
Minimum - Maximum0.0 - 0.01970059
Average (Standard dev.)0.00154661 (±0.00334466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin151551
Dimensions250250178
Spacing250250178
CellA: 725.0 Å / B: 725.0 Å / C: 516.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z250250178
origin x/y/z0.0000.0000.000
length x/y/z725.000725.000516.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS151551
NC/NR/NS250250178
D min/max/mean0.0000.0200.002

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Supplemental data

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Sample components

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Entire : Helically organized porcine blood coagulation Factor VIII bound t...

EntireName: Helically organized porcine blood coagulation Factor VIII bound to a lipid nanotube
Components
  • Sample: Helically organized porcine blood coagulation Factor VIII bound to a lipid nanotube
  • Protein or peptide: Blood coagulation Factor VIII
  • Ligand: lipid nanotubes

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Supramolecule #1000: Helically organized porcine blood coagulation Factor VIII bound t...

SupramoleculeName: Helically organized porcine blood coagulation Factor VIII bound to a lipid nanotube
type: sample / ID: 1000 / Oligomeric state: dimer / Number unique components: 2
Molecular weightExperimental: 170 KDa / Theoretical: 170 KDa / Method: SDS gel and amino acid sequence

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Macromolecule #1: Blood coagulation Factor VIII

MacromoleculeName: Blood coagulation Factor VIII / type: protein_or_peptide / ID: 1 / Name.synonym: Hemophilia A factor
Details: Recombinant porcine Factor VIII lacking the B domain was bound to lipid nanotubes and helically organized for structure analysis by cryo-EM.
Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Sus scrofa (pig) / synonym: PIG / Cell: blood
Molecular weightExperimental: 170 KDa / Theoretical: 170 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: BHK cells

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Macromolecule #2: lipid nanotubes

MacromoleculeName: lipid nanotubes / type: ligand / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCL, 5 mM CaCl2
StainingType: NEGATIVE
Details: samples are adsorbed on quantifoil grids and flash frozen in liquid ethane
GridDetails: carbon coated quantifoil grids Q2x2 were glow discharged for 100 second
VitrificationCryogen name: ETHANE / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK IV / Method: blot for 3.5 seconds, force 1 before plunging
DetailsThe protein and lipid are mixed in 1:1 ration and incubated for 15 minutes

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.004 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 99 K
DateSep 11, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 300 / Average electron dose: 16 e/Å2

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Image processing

CTF correctionDetails: each particle set
Final reconstructionApplied symmetry - Helical parameters - Δz: 36 Å
Applied symmetry - Helical parameters - Δ&Phi: 35.5 °
Applied symmetry - Helical parameters - Axial symmetry: C5 (5 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 15.5 Å / Resolution method: OTHER
DetailsDescribed in supplemental materials and references

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Atomic model buiding 1

Initial modelPDB ID:

3cdz


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: UCSF Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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