+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8288 | |||||||||
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Title | Putative tetramer of human CPAP (residues 897-1338) | |||||||||
Map data | Contour level of 0.031 using Chimera | |||||||||
Sample |
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Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 23.0 Å | |||||||||
Authors | Alvarez-Cabrera AL / Delgado S / Gil D / Mortuza G / Montoya G / Sorzano CO / Tang TK / Carazo JM | |||||||||
Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8288.map.gz | 126.4 KB | EMDB map data format | |
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Header (meta data) | emd-8288-v30.xml emd-8288.xml | 12.3 KB 12.3 KB | Display Display | EMDB header |
Images | emd_8288.png | 32.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8288 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8288 | HTTPS FTP |
-Validation report
Summary document | emd_8288_validation.pdf.gz | 78.4 KB | Display | EMDB validaton report |
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Full document | emd_8288_full_validation.pdf.gz | 77.5 KB | Display | |
Data in XML | emd_8288_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8288 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8288 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8288.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Contour level of 0.031 using Chimera | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.42 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Putative homotetramer of human CPAP (residues 897-1338)
Entire | Name: Putative homotetramer of human CPAP (residues 897-1338) |
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Components |
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-Supramolecule #1: Putative homotetramer of human CPAP (residues 897-1338)
Supramolecule | Name: 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) |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) Recombinant plasmid: pST66Trc2-His (is a pET3a modified plasmid) |
Molecular weight | Experimental: 208 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 Component:
Details: TCEP added to the buffer was prepared fresh | |||||||||||||||
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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 |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Image recording | Film 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 beam | Acceleration voltage: 200 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |