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- PDB-7r03: Neurofibromin occluded conformation -

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Basic information

Entry
Database: PDB / ID: 7r03
TitleNeurofibromin occluded conformation
ComponentsIsoform I of Neurofibromin
KeywordsSIGNALING PROTEIN / Signalling protein / GAP / Neurofibromatosis type I / Homodimer
Function / homology
Function and homology information


positive regulation of mast cell apoptotic process / negative regulation of Rac protein signal transduction / regulation of glial cell differentiation / observational learning / Schwann cell migration / negative regulation of Schwann cell migration / vascular associated smooth muscle cell migration / amygdala development / negative regulation of mast cell proliferation / gamma-aminobutyric acid secretion, neurotransmission ...positive regulation of mast cell apoptotic process / negative regulation of Rac protein signal transduction / regulation of glial cell differentiation / observational learning / Schwann cell migration / negative regulation of Schwann cell migration / vascular associated smooth muscle cell migration / amygdala development / negative regulation of mast cell proliferation / gamma-aminobutyric acid secretion, neurotransmission / mast cell apoptotic process / Schwann cell proliferation / vascular associated smooth muscle cell proliferation / mast cell proliferation / glutamate secretion, neurotransmission / negative regulation of Schwann cell proliferation / negative regulation of leukocyte migration / negative regulation of neurotransmitter secretion / negative regulation of vascular associated smooth muscle cell migration / positive regulation of adenylate cyclase activity / forebrain morphogenesis / hair follicle maturation / regulation of cell-matrix adhesion / regulation of blood vessel endothelial cell migration / smooth muscle tissue development / camera-type eye morphogenesis / cell communication / negative regulation of oligodendrocyte differentiation / sympathetic nervous system development / myeloid leukocyte migration / peripheral nervous system development / phosphatidylcholine binding / myelination in peripheral nervous system / metanephros development / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / negative regulation of Ras protein signal transduction / phosphatidylethanolamine binding / collagen fibril organization / regulation of bone resorption / regulation of long-term synaptic potentiation / neural tube development / endothelial cell proliferation / forebrain astrocyte development / artery morphogenesis / regulation of postsynapse organization / pigmentation / negative regulation of neuroblast proliferation / adrenal gland development / negative regulation of cell-matrix adhesion / regulation of synaptic transmission, GABAergic / negative regulation of protein import into nucleus / regulation of GTPase activity / spinal cord development / Rac protein signal transduction / negative regulation of osteoclast differentiation / negative regulation of endothelial cell proliferation / oligodendrocyte differentiation / RAS signaling downstream of NF1 loss-of-function variants / negative regulation of astrocyte differentiation / extrinsic apoptotic signaling pathway via death domain receptors / neuroblast proliferation / negative regulation of MAPK cascade / regulation of angiogenesis / Schwann cell development / negative regulation of stem cell proliferation / negative regulation of fibroblast proliferation / negative regulation of MAP kinase activity / extrinsic apoptotic signaling pathway in absence of ligand / skeletal muscle tissue development / positive regulation of vascular associated smooth muscle cell proliferation / positive regulation of endothelial cell proliferation / phosphatidylinositol 3-kinase/protein kinase B signal transduction / extracellular matrix organization / GTPase activator activity / negative regulation of angiogenesis / regulation of ERK1 and ERK2 cascade / osteoclast differentiation / positive regulation of GTPase activity / liver development / negative regulation of cell migration / stem cell proliferation / long-term synaptic potentiation / negative regulation of protein kinase activity / wound healing / regulation of long-term neuronal synaptic plasticity / visual learning / brain development / cerebral cortex development / cognition / osteoblast differentiation / protein import into nucleus / Regulation of RAS by GAPs / positive regulation of neuron apoptotic process / MAPK cascade / presynapse / heart development / cellular response to heat / actin cytoskeleton organization / fibroblast proliferation / regulation of gene expression
Similarity search - Function
: / PH domain-like / Ras GTPase-activating protein / Ras GTPase-activating protein, conserved site / Ras GTPase-activating proteins domain signature. / GTPase-activator protein for Ras-like GTPase / Ras GTPase-activating proteins profile. / GTPase-activator protein for Ras-like GTPases / Ras GTPase-activating domain / Divergent CRAL/TRIO domain ...: / PH domain-like / Ras GTPase-activating protein / Ras GTPase-activating protein, conserved site / Ras GTPase-activating proteins domain signature. / GTPase-activator protein for Ras-like GTPase / Ras GTPase-activating proteins profile. / GTPase-activator protein for Ras-like GTPases / Ras GTPase-activating domain / Divergent CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) / CRAL-TRIO lipid binding domain / CRAL-TRIO lipid binding domain superfamily / Rho GTPase activation protein / PH-like domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChaker-Margot, M. / Scheffzek, K. / Maier, T.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP) Switzerland
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of activation of the tumor suppressor protein neurofibromin.
Authors: Malik Chaker-Margot / Sebastiaan Werten / Theresia Dunzendorfer-Matt / Stefan Lechner / Angela Ruepp / Klaus Scheffzek / Timm Maier /
Abstract: Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and ...Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.
History
DepositionFeb 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Apr 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform I of Neurofibromin
B: Isoform I of Neurofibromin


Theoretical massNumber of molelcules
Total (without water)634,8222
Polymers634,8222
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Isoform I of Neurofibromin / Neurofibromatosis-related protein NF-1


Mass: 317410.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NF1 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P21359

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Neurofibromin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 640 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 93 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF 200

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19rc4_4035refinement
PHENIX1.19rc4_4035refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
7Coot0.9.4model fitting
12cryoSPARC2.143D reconstruction
13PHENIX1.19model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 360000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
16V65

6v65

B6V651
22E2X

2e2x

A2E2X2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 275.09 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005436339
ELECTRON MICROSCOPYf_angle_d1.087849252
ELECTRON MICROSCOPYf_chiral_restr0.05775778
ELECTRON MICROSCOPYf_plane_restr0.00836183
ELECTRON MICROSCOPYf_dihedral_angle_d6.81444739

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