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- EMDB-8288: Putative tetramer of human CPAP (residues 897-1338) -

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Basic information

Entry
Database: EMDB / ID: EMD-8288
TitlePutative tetramer of human CPAP (residues 897-1338)
Map dataContour level of 0.031 using Chimera
Sample
  • Complex: Putative homotetramer of human CPAP (residues 897-1338)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsAlvarez-Cabrera AL / Delgado S / Gil D / Mortuza G / Montoya G / Sorzano CO / Tang TK / Carazo JM
CitationJournal: Front Mol Biosci / Year: 2017
Title: Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome.
Authors: Ana L Alvarez-Cabrera / Sandra Delgado / David Gil-Carton / Gulnahar B Mortuza / Guillermo Montoya / Carlos O S Sorzano / Tang K Tang / Jose M Carazo /
Abstract: Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP ...Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of , using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of CPAP (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis.
History
DepositionJul 13, 2016-
Header (metadata) releaseSep 14, 2016-
Map releaseSep 14, 2016-
UpdateApr 18, 2018-
Current statusApr 18, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.031
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.031
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8288.png

Downloads & links

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Map

FileDownload / File: emd_8288.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationContour level of 0.031 using Chimera
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.42 Å/pix.
x 60 pix.
= 265.2 Å		4.42 Å/pix.
x 60 pix.
= 265.2 Å		4.42 Å/pix.
x 60 pix.
= 265.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.031 / Movie #1: 0.031
Minimum - Maximum-0.034058508 - 0.10883167
Average (Standard dev.)0.0011130079 (±0.007355001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-30-30
Dimensions606060
Spacing606060
CellA=B=C: 265.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.424.424.42
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z265.200265.200265.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-128-128-128
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS-30-30-30
NC/NR/NS606060
D min/max/mean-0.0340.1090.001

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Supplemental data

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Sample components

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Entire : Putative homotetramer of human CPAP (residues 897-1338)

EntireName: Putative homotetramer of human CPAP (residues 897-1338)
Components
  • Complex: Putative homotetramer of human CPAP (residues 897-1338)

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Supramolecule #1: Putative homotetramer of human CPAP (residues 897-1338)

SupramoleculeName: Putative homotetramer of human CPAP (residues 897-1338)
type: complex / ID: 1 / Parent: 0
Details: Human CPAP (residues 897-1338) expresed in E. coli and purified by Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Recombinant plasmid: pST66Trc2-His (is a pET3a modified plasmid)
Molecular weightExperimental: 208 KDa

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
300.0 mMNaClSodiun chloride
0.25 mMC9H15O6PTCEP
10.0 %C3H8O3Glycerol

Details: TCEP added to the buffer was prepared fresh
StainingType: NEGATIVE / Material: Uranyl Formate (0.75%)
GridModel: Quantifoil. Formvar/Carbon / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: CONTINUOUS / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: FORMVAR / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
DetailsThis human CPAP construct includes residues 897-1338

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 9.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3048
CTF correctionSoftware - Name: Xmipp (ver. 3.1)
Startup modelType of model: OTHER
Details: RANSAC algorithm (Bioinformatics. 2014 Oct 15;30(20):2891-8)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Xmipp (ver. 3.1) / Software - details: Reconstruct Significant algorithm / Number images used: 3048
Initial angle assignmentType: OTHER / Software - Name: Xmipp (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.2) / Software - details: Relion (accessed from Xmipp3.1)
Final 3D classificationSoftware - Name: RELION (ver. 1.2) / Software - details: Relion (accessed from Xmipp3.1)

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