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- EMDB-20667: Biochemical and structural analysis of the Neurofibromin (NF1) pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-20667
TitleBiochemical and structural analysis of the Neurofibromin (NF1) protein reveals high-affinity dimer formation
Map dataNegative Stain Reconstruction
Sample
  • Complex: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3).
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 18.5 Å
AuthorsJuneja 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
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney DiseaseHHSN261200800001E United States
Department of Energy (United States)DE-AC05-00OR22725 United States
CitationJournal: 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
DepositionAug 30, 2019-
Header (metadata) releaseJan 8, 2020-
Map releaseJan 8, 2020-
UpdateFeb 5, 2020-
Current statusFeb 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20667.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative Stain Reconstruction
Voxel sizeX=Y=Z: 1.77 Å
Density
Contour LevelBy EMDB: 0.013 / Movie #1: 0.01
Minimum - Maximum-0.0412064 - 0.04596241
Average (Standard dev.)0.0000663630 (±0.00220435)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 573.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z573.480573.480573.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean-0.0410.0460.000

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Supplemental data

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Sample components

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Entire : Neurofibromin is the protein product of the NF1 gene Human NF1 is...

EntireName: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3).
Components
  • Complex: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3).

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Supramolecule #1: Neurofibromin is the protein product of the NF1 gene Human NF1 is...

SupramoleculeName: Neurofibromin is the protein product of the NF1 gene Human NF1 isoform 2 (NM_000267.3).
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant strain: Tni-FNL
Molecular weightTheoretical: 317 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

Concentration0.01 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HclTrisTris-HclTris
300.0 mMNaClSodium chlorideSodium chloride
5.0 mMC9H15O6PTCEP

Details: in 20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 5 mM TCEP
StainingType: NEGATIVE / Material: uranyl formate / Details: 0.75% w/v uranyl formate, pH 4.5, for 30 sec
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
DetailsSample was mono disperse

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 41951 / Details: EMAN2 e2boxer
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 18.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPHIRE (ver. EMAN 2.21a final) / Number images used: 30588
DetailsGatan Ultra Scan camera

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