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- EMDB-6011: Electron microscopy of Calsyntenin-3 tetramer conformation I -

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Basic information

Entry
Database: EMDB / ID: EMD-6011
TitleElectron microscopy of Calsyntenin-3 tetramer conformation I
Map dataCstn-3 High Molecular Weight (HMW) tetramer conformation I
Sample
  • Sample: Cstn-3 Tetramer (High Molecular Weight)
  • Protein or peptide: Calsyntenin-3
KeywordsSynaptic organizer / trans-synaptic bridge
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsLu Z / Wang Y / Chen F / Tong F / Reddy MVVVS / Luo L / Seshadrinathan S / Zhang L / Holthauzen LMF / Craig AM ...Lu Z / Wang Y / Chen F / Tong F / Reddy MVVVS / Luo L / Seshadrinathan S / Zhang L / Holthauzen LMF / Craig AM / Ren G / Rudenko G
CitationJournal: J Biol Chem / Year: 2014
Title: Calsyntenin-3 molecular architecture and interaction with neurexin 1α.
Authors: Zhuoyang Lu / Yun Wang / Fang Chen / Huimin Tong / M V V V Sekhar Reddy / Lin Luo / Suchithra Seshadrinathan / Lei Zhang / Luis Marcelo F Holthauzen / Ann Marie Craig / Gang Ren / Gabby Rudenko /
Abstract: Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. ...Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca(2+), and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca(2+) and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function.
History
DepositionAug 3, 2014-
Header (metadata) releaseAug 20, 2014-
Map releaseSep 2, 2015-
UpdateSep 2, 2015-
Current statusSep 2, 2015Processing site: RCSB / 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

400_6011.gif

Downloads & links

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Map

FileDownload / File: emd_6011.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCstn-3 High Molecular Weight (HMW) tetramer conformation I
Voxel sizeX=Y=Z: 1.88 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.35349503 - 1.49966288
Average (Standard dev.)0.00192437 (±0.19413492)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-34-34-35
Dimensions128128128
Spacing128128128
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.881.881.88
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z240.640240.640240.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-34-34-35
NC/NR/NS128128128
D min/max/mean-0.3531.5000.002

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Supplemental data

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Sample components

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Entire : Cstn-3 Tetramer (High Molecular Weight)

EntireName: Cstn-3 Tetramer (High Molecular Weight)
Components
  • Sample: Cstn-3 Tetramer (High Molecular Weight)
  • Protein or peptide: Calsyntenin-3

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Supramolecule #1000: Cstn-3 Tetramer (High Molecular Weight)

SupramoleculeName: Cstn-3 Tetramer (High Molecular Weight) / type: sample / ID: 1000 / Oligomeric state: Tetramer / Number unique components: 1
Molecular weightTheoretical: 352 KDa
Method: Calculated from amino acid content without N-linked glycosylation

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Macromolecule #1: Calsyntenin-3

MacromoleculeName: Calsyntenin-3 / type: protein_or_peptide / ID: 1 / Name.synonym: Cstn-3 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: neuron / Location in cell: Synapse
Molecular weightTheoretical: 88 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant strain: BTI-TN-5B1-4 / Recombinant cell: High Five

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8 / Details: 25 mM Tris, pH 8.0, 120 mM NaCl, 5 mM CaCl2
StainingType: NEGATIVE
Details: An aliquot (~4 uL) of sample was placed on a grid. After ~1 min incubation, excess solution was blotted with filter paper, and the grid stained for ~1 min by submersion in one drop (~35 uL) ...Details: An aliquot (~4 uL) of sample was placed on a grid. After ~1 min incubation, excess solution was blotted with filter paper, and the grid stained for ~1 min by submersion in one drop (~35 uL) of 1% w/v uranyl formate (UF) on Parafilm before being nitrogen-air-dried at room temperature.
GridDetails: thin-carbon-coated 200 mesh copper grid (CF200-Cu, EMS), glow-discharged 15 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 0.6 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 125000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification.
DateDec 11, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 639 / Average electron dose: 200 e/Å2

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Image processing

CTF correctionDetails: ctffind3
Final two d classificationNumber classes: 150
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: EMAN, IPET / Number images used: 6699
DetailsThe ab initio initial model was reconstructed by individual particle electron tomography, in which a single molecule of LMW particle was imaged from a tilt series (angle range from -66 to +66 degrees, in steps of 1.5 degree).

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