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Yorodumi- EMDB-20667: Biochemical and structural analysis of the Neurofibromin (NF1) pr... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20667 | |||||||||
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Title | Biochemical and structural analysis of the Neurofibromin (NF1) protein reveals high-affinity dimer formation | |||||||||
Map data | Negative Stain Reconstruction | |||||||||
Sample |
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Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 18.5 Å | |||||||||
Authors | Juneja P / Sherekar M / Esposito D / Han SW / Ghirlando R / Messing S / Drew M / Niel OH / Stanley C / Bhowmik D ...Juneja P / Sherekar M / Esposito D / Han SW / Ghirlando R / Messing S / Drew M / Niel OH / Stanley C / Bhowmik D / Ramanathan A / Subramaniam S / Nissley D / Gillette W / McCormick F | |||||||||
Funding support | United States, 2 items
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Citation | Journal: J Biol Chem / Year: 2020 Title: Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer. Authors: Mukul Sherekar / Sae-Won Han / Rodolfo Ghirlando / Simon Messing / Matthew Drew / Dana Rabara / Timothy Waybright / Puneet Juneja / Hugh O'Neill / Christopher B Stanley / Debsindhu Bhowmik / ...Authors: Mukul Sherekar / Sae-Won Han / Rodolfo Ghirlando / Simon Messing / Matthew Drew / Dana Rabara / Timothy Waybright / Puneet Juneja / Hugh O'Neill / Christopher B Stanley / Debsindhu Bhowmik / Arvind Ramanathan / Sriram Subramaniam / Dwight V Nissley / William Gillette / Frank McCormick / Dominic Esposito / Abstract: Neurofibromin is a tumor suppressor encoded by the gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in lead to aberrant signaling through the RAS-mitogen-activated ...Neurofibromin is a tumor suppressor encoded by the gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains The reconstituted neurofibromin was capable of GTPase activation , and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell. | |||||||||
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_20667.map.gz | 7.1 MB | EMDB map data format | |
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Header (meta data) | emd-20667-v30.xml emd-20667.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | emd_20667.png | 29.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20667 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20667 | HTTPS FTP |
-Validation report
Summary document | emd_20667_validation.pdf.gz | 78.4 KB | Display | EMDB validaton report |
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Full document | emd_20667_full_validation.pdf.gz | 77.5 KB | Display | |
Data in XML | emd_20667_validation.xml.gz | 496 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20667 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20667 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_20667.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Negative Stain Reconstruction | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.77 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 : Neurofibromin is the protein product of the NF1 gene Human NF1 is...
Entire | Name: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3). |
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Components |
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-Supramolecule #1: Neurofibromin is the protein product of the NF1 gene Human NF1 is...
Supramolecule | Name: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3). type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) / Recombinant strain: Tni-FNL |
Molecular weight | Theoretical: 317 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
Details: in 20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 5 mM TCEP | ||||||||||||
Staining | Type: NEGATIVE / Material: uranyl formate / Details: 0.75% w/v uranyl formate, pH 4.5, for 30 sec | ||||||||||||
Grid | Model: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | ||||||||||||
Details | Sample was mono disperse |
-Electron microscopy
Microscope | FEI TECNAI 12 |
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Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |