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- EMDB-2944: Structural characterization of the Olfactomedin-1 disulfide-linke... -

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Basic information

Entry
Database: EMDB / ID: EMD-2944
TitleStructural characterization of the Olfactomedin-1 disulfide-linked tetramer
Map dataSubtomogram of an Olfactomedin-1 tetramer
Sample
  • Sample: Olfactomedin-1 disulfide-linked tetramer
  • Organelle or cellular component: Olfactomedin-1
KeywordsOlfactomedin-1 (Olfm1) / neurobiology / tetramer / X-ray crystallography / small-angle X-ray scattering (SAXS) / electron tomography / analytical ultracentrifugation (AUC) / coiled coil / calcium / disulfide
Biological speciesMus musculus (house mouse)
Methodelectron tomography / negative staining
AuthorsSharp TH / Pronker MF / Bos TG / Thies-Weesie DM / Janssen BJC
CitationJournal: J Biol Chem / Year: 2015
Title: Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure.
Authors: Matti F Pronker / Trusanne G A A Bos / Thomas H Sharp / Dominique M E Thies-Weesie / Bert J C Janssen /
Abstract: Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system ...Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the "V" is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1(coil-Olf)) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members.
History
DepositionMar 18, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseNov 18, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2944.png

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Map

FileDownload / File: emd_2944.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram of an Olfactomedin-1 tetramer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.57 Å/pix.
x 100 pix.
= 457. Å		4.57 Å/pix.
x 100 pix.
= 457. Å		4.57 Å/pix.
x 100 pix.
= 457. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.57 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-6.57791138 - 9.39217281
Average (Standard dev.)0.01143731 (±0.31603697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 457.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.574.574.57
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z457.000457.000457.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS100100100
D min/max/mean-6.5789.3920.011

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Supplemental data

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Sample components

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Entire : Olfactomedin-1 disulfide-linked tetramer

EntireName: Olfactomedin-1 disulfide-linked tetramer
Components
  • Sample: Olfactomedin-1 disulfide-linked tetramer
  • Organelle or cellular component: Olfactomedin-1

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Supramolecule #1000: Olfactomedin-1 disulfide-linked tetramer

SupramoleculeName: Olfactomedin-1 disulfide-linked tetramer / type: sample / ID: 1000 / Oligomeric state: Tetramer / Number unique components: 1
Molecular weightExperimental: 242 KDa / Theoretical: 256 KDa / Method: AUC

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Supramolecule #1: Olfactomedin-1

SupramoleculeName: Olfactomedin-1 / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Olfm1, noelin, pancortin
Details: Purified recombinant full-length glycoslated Olfactomedin-1 tetramers were negatively stained with uranyl formate.
Number of copies: 1 / Oligomeric state: Tetramer / Recombinant expression: Yes
Ref INTERPRO0: IPR022082
Ref INTERPRO0: IPR003112
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse / Tissue: brain / Cell: neuron / Organelle: Secretory pathway / Location in cell: Secreted (ER/golgi/extracellular)
Molecular weightExperimental: 242 KDa / Theoretical: 256 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293ES / Recombinant plasmid: pUPE107.03

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.065 mg/mL
BufferpH: 7.5 / Details: 150 mM NaCl, 20 mM HEPES
StainingType: NEGATIVE
Details: Full-length Olfm1 was adsorbed to grids for 30 sec. Grids were briefly washed with water and then stained for 30 sec with a freshly prepared filtered 2% uranyl formate solution.
GridDetails: Carbon-coated mesh copper grids glow discharged for 15 sec
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 30,000 times magnification
DetailsWeak beam illumination
DateFeb 12, 2015
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 58 / Bits/pixel: 32
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -58 ° / Tilt series - Axis1 - Max angle: 58 ° / Tilt series - Axis1 - Angle increment: 2 °
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsTilt series were collected from -58 to 58 degrees in 2 degree increments. The first and last image was discarded. CTF correction was performed on each projection
Final reconstructionAlgorithm: OTHER / Software - Name: IMOD, EMAN2
Details: Sub-tomogram particles were manually picked using e2spt_boxer.py from EMAN2. Each particle was normalized and masked with a sharp spherical mask to remove background density not associated ...Details: Sub-tomogram particles were manually picked using e2spt_boxer.py from EMAN2. Each particle was normalized and masked with a sharp spherical mask to remove background density not associated with the protein. Particles were then filtered to 20 A with a low-pass Gaussian filter, before a tight mask was applied to the remaining density using e2proc3d.py from EMAN2.
Number images used: 57
CTF correctionDetails: IMOD, each tilt image

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Atomic model buiding 1

Initial modelPDB ID:

5amo


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsEach coiled-coil dimer of the tetramer was fitted as a rigid body.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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