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- EMDB-3026: Electron cryo-microscopy of porcine Factor VIII bound to lipid na... -

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Basic information

Entry
Database: EMDB / ID: EMD-3026
TitleElectron cryo-microscopy of porcine Factor VIII bound to lipid nanotubes and single particle tomography reconstruction
Map dataSPT reconstruction from tomograms of negatively stained porcine FVIII bound to LNT in a helical organization. The volume is low pass filter to 20 Angstroms in EMAN2
Sample
  • Sample: Helically organized porcine blood coagulation Factor VIII bound to a lipid nanotube
  • Protein or peptide: Blood coagulation Factor VIII
  • Ligand: LIPID NANOTUBE
KeywordsBlood coagulation factor VIII / Lipid nanotubes / Cryo-electron microscopy / Membrane-bound organization / Single particle tomography reconstruction
Biological speciesSus scrofa (pig) / unidentified (others)
Methodsubtomogram averaging / negative staining / Resolution: 21.0 Å
AuthorsDalm D / Galaz-Montoya JG / Miller JL / Grushin K / Villalobos A / Koyfman AY / Schmid MF / Stoilova-McPhie S
CitationJournal: J Struct Biol / Year: 2015
Title: Single particle tomography in EMAN2.
Authors: Jesús G Galaz-Montoya / John Flanagan / Michael F Schmid / Steven J Ludtke /
Abstract: Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally ...Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally heterogeneous macromolecules. It can also provide direct observation (without labeling or staining) of complexes inside cells at nanometer resolution. The development of computational methods and tools for SPT remains an area of active research. Here we present the EMAN2.1 SPT toolbox, which offers a full SPT processing pipeline, from particle picking to post-alignment analysis of subtomogram averages, automating most steps. Different algorithm combinations can be applied at each step, providing versatility and allowing for procedural cross-testing and specimen-specific strategies. Alignment methods include all-vs-all, binary tree, iterative single-model refinement, multiple-model refinement, and self-symmetry alignment. An efficient angular search, Graphic Processing Unit (GPU) acceleration and both threaded and distributed parallelism are provided to speed up processing. Finally, automated simulations, per particle reconstruction of subtiltseries, and per-particle Contrast Transfer Function (CTF) correction have been implemented. Processing examples using both real and simulated data are shown for several structures.
History
DepositionMay 27, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseAug 12, 2015-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 100
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 100
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3026.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSPT reconstruction from tomograms of negatively stained porcine FVIII bound to LNT in a helical organization. The volume is low pass filter to 20 Angstroms in EMAN2
Voxel sizeX=Y=Z: 2.9 Å
Density
Contour LevelBy AUTHOR: 30.0 / Movie #1: 100
Minimum - Maximum0.0 - 699.017517089999956
Average (Standard dev.)30.404388430000001 (±84.800666809999996)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 278.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z278.400278.400278.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean0.000699.01830.404

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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 NANOTUBE

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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
Details: The protein was monodisperse in solution and bound to the lipid nanotube in 1:1 w/w ratio to achieve helical organization
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: 4 / Oligomeric state: 2 / 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

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Macromolecule #2: LIPID NANOTUBE

MacromoleculeName: LIPID NANOTUBE / 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
Processingsubtomogram averaging
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: The pFVIII-LNT ehlcial tubes were absorbed on amorphous carbon and stained with 1% Uranyl acetate
GridDetails: Evaporated carbon on mice was floated on 300 mesh copper grids, dried, glow discharged for 10 seconds
VitrificationCryogen name: NONE / Instrument: OTHER
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: -0.0025 µm / Nominal defocus min: -0.001 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: 2 ° / Tilt series - Axis1 - Max angle: 60 °
DateOct 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20 e/Å2

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Image processing

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: 21.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number subtomograms used: 756
Detailsdescribed in details in the submitted references

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Atomic model buiding 1

Initial modelPDB ID:

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

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