Journal: 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
Deposition
Jul 11, 2016
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Header (metadata) release
Jul 20, 2016
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Map release
Jul 20, 2016
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Update
Apr 18, 2018
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Current status
Apr 18, 2018
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_8283.map.gz / Format: CCP4 / Size: 489.3 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Contour level of 0.147 when visualized with chimera
Voxel size
X=Y=Z: 1 Å
Density
Contour Level
By AUTHOR: 0.147 / Movie #1: 0.147
Minimum - Maximum
-0.25500903 - 0.35606775
Average (Standard dev.)
0.003670858 (±0.035643324)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
50
50
50
Spacing
50
50
1
Cell
A: 50.0 Å / B: 50.0 Å / C: 1.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1
1
1
M x/y/z
50
50
1
origin x/y/z
0.000
0.000
0.000
length x/y/z
50.000
50.000
1.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-128
-128
-128
NX/NY/NZ
256
256
256
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
50
50
50
D min/max/mean
-0.255
0.356
0.004
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Supplemental data
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Sample components
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Entire : Putative homodimer of human CPAP (residues 897-1338)
Entire
Name: Putative homodimer of human CPAP (residues 897-1338)
Components
Complex: Putative homodimer of human CPAP (residues 897-1338)
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Supramolecule #1: Putative homodimer of human CPAP (residues 897-1338)
Supramolecule
Name: Putative homodimer 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)
Details: TCEP added to the buffer was prepared fresh
Staining
Type: NEGATIVE / Material: Uranyl Formate (0.75%)
Grid
Model: 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
Details
This human CPAP construct includes residues 897-1338
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Electron microscopy
Microscope
JEOL 1230
Image recording
Film or detector model: GATAN ORIUS SC1000 (4k x 2.7k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 9.0 e/Å2
Electron beam
Acceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron optics
Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm
+
Image processing
Particle selection
Number selected: 36699
CTF correction
Software - Name: Xmipp (ver. 3.1)
Startup model
Type of model: OTHER Details: RANSAC algorithm (Bioinformatics. 2014 Oct 15;30(20):2891-8)
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.2) / Software - details: Relion (accessed from Xmipp3.1) / Number images used: 36699
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