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- EMDB-14219: Composite map of the open conformation of neurofibromin -

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Basic information

Entry
Database: EMDB / ID: EMD-14219
TitleComposite map of the open conformation of neurofibromin
Map dataComposite map of neurofibromin in open conformation
Sample
  • Complex: Homodimer of Neurofibromin
    • Protein or peptide: Isoform I of Neurofibromin
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
KeywordsSignalling protein / Homodimer / Neurofibromatosis / GAP / SIGNALING PROTEIN
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 / peripheral nervous system development / sympathetic nervous system development / myeloid leukocyte migration / myelination in peripheral nervous system / phosphatidylcholine binding / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / metanephros development / negative regulation of Ras protein signal transduction / phosphatidylethanolamine binding / regulation of bone resorption / collagen fibril organization / regulation of long-term synaptic potentiation / neural tube development / endothelial cell proliferation / forebrain astrocyte development / artery morphogenesis / pigmentation / regulation of postsynapse organization / negative regulation of neuroblast proliferation / negative regulation of protein import into nucleus / adrenal gland development / regulation of synaptic transmission, GABAergic / negative regulation of cell-matrix adhesion / 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 / extrinsic apoptotic signaling pathway in absence of ligand / negative regulation of fibroblast proliferation / negative regulation of MAP kinase activity / positive regulation of vascular associated smooth muscle cell proliferation / skeletal muscle tissue development / positive regulation of endothelial cell proliferation / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of ERK1 and ERK2 cascade / GTPase activator activity / extracellular matrix organization / negative regulation of angiogenesis / osteoclast differentiation / liver development / positive regulation of GTPase activity / 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 / brain development / visual learning / cerebral cortex development / cognition / osteoblast differentiation / Regulation of RAS by GAPs / protein import into nucleus / positive regulation of neuron apoptotic process / MAPK cascade / heart development / presynapse / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsChaker-Margot M / Scheffzek K
Funding support Switzerland, 1 items
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, 2022-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14219.png

Downloads & links

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Map

FileDownload / File: emd_14219.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of neurofibromin in open conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 512 pix.
= 541.696 Å		1.06 Å/pix.
x 512 pix.
= 541.696 Å		1.06 Å/pix.
x 512 pix.
= 541.696 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.058 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-20.737380999999999 - 52.847250000000003
Average (Standard dev.)0.0023240442 (±0.9975857)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 541.696 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Neurofibromin in open conformation (unmasked)

Fileemd_14219_additional_1.map
AnnotationNeurofibromin in open conformation (unmasked)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Neurofibromin in open conformation (masked refinement on half 1)

Fileemd_14219_additional_2.map
AnnotationNeurofibromin in open conformation (masked refinement on half 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Neurofibromin in open conformation (masked refinement on half 2)

Fileemd_14219_additional_3.map
AnnotationNeurofibromin in open conformation (masked refinement on half 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimer of Neurofibromin

EntireName: Homodimer of Neurofibromin
Components
  • Complex: Homodimer of Neurofibromin
    • Protein or peptide: Isoform I of Neurofibromin
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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Supramolecule #1: Homodimer of Neurofibromin

SupramoleculeName: Homodimer of Neurofibromin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 640 kDa/nm

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Macromolecule #1: Isoform I of Neurofibromin

MacromoleculeName: Isoform I of Neurofibromin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 317.410812 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAAHRPVEWV QAVVSRFDEQ LPIKTGQQNT HTKVSTEHNK ECLINISKYK FSLVISGLTT ILKNVNNMRI FGEAAEKNLY LSQLIILDT LEKCLAGQPK DTMRLDETML VKQLLPEICH FLHTCREGNQ HAAELRNSAS GVLFSLSCNN FNAVFSRIST R LQELTVCS ...String:
MAAHRPVEWV QAVVSRFDEQ LPIKTGQQNT HTKVSTEHNK ECLINISKYK FSLVISGLTT ILKNVNNMRI FGEAAEKNLY LSQLIILDT LEKCLAGQPK DTMRLDETML VKQLLPEICH FLHTCREGNQ HAAELRNSAS GVLFSLSCNN FNAVFSRIST R LQELTVCS EDNVDVHDIE LLQYINVDCA KLKRLLKETA FKFKALKKVA QLAVINSLEK AFWNWVENYP DEFTKLYQIP QT DMAECAE KLFDLVDGFA ESTKRKAAVW PLQIILLILC PEIIQDISKD VVDENNMNKK LFLDSLRKAL AGHGGSRQLT ESA AIACVK LCKASTYINW EDNSVIFLLV QSMVVDLKNL LFNPSKPFSR GSQPADVDLM IDCLVSCFRI SPHNNQHFKI CLAQ NSPST FHYVLVNSLH RIITNSALDW WPKIDAVYCH SVELRNMFGE TLHKAVQGCG AHPAIRMAPS LTFKEKVTSL KFKEK PTDL ETRSYKYLLL SMVKLIHADP KLLLCNPRKQ GPETQGSTAE LITGLVQLVP QSHMPEIAQE AMEALLVLHQ LDSIDL WNP DAPVETFWEI SSQMLFYICK KLTSHQMLSS TEILKWLREI LICRNKFLLK NKQADRSSCH FLLFYGVGCD IPSSGNT SQ MSMDHEELLR TPGASLRKGK GNSSMDSAAG CSGTPPICRQ AQTKLEVALY MFLWNPDTEA VLVAMSCFRH LCEEADIR C GVDEVSVHNL LPNYNTFMEF ASVSNMMSTG RAALQKRVMA LLRRIEHPTA GNTEAWEDTH AKWEQATKLI LNYPKAKME DGQAAESLHK TIVKRRMSHV SGGGSIDLSD TDSLQEWINM TGFLCALGGV CLQQRSNSGL ATYSPPMGPV SERKGSMISV MSSEGNADT PVSKFMDRLL SLMVCNHEKV GLQIRTNVKD LVGLELSPAL YPMLFNKLKN TISKFFDSQG QVLLTDTNTQ F VEQTIAIM KNLLDNHTEG SSEHLGQASI ETMMLNLVRY VRVLGNMVHA IQIKTKLCQL VEVMMARRDD LSFCQEMKFR NK MVEYLTD WVMGTSNQAA DDDVKCLTRD LDQASMEAVV SLLAGLPLQP EEGDGVELME AKSQLFLKYF TLFMNLLNDC SEV EDESAQ TGGRKRGMSR RLASLRHCTV LAMSNLLNAN VDSGLMHSIG LGYHKDLQTR ATFMEVLTKI LQQGTEFDTL AETV LADRF ERLVELVTMM GDQGELPIAM ALANVVPCSQ WDELARVLVT LFDSRHLLYQ LLWNMFSKEV ELADSMQTLF RGNSL ASKI MTFCFKVYGA TYLQKLLDPL LRIVITSSDW QHVSFEVDPT RLEPSESLEE NQRNLLQMTE KFFHAIISSS SEFPPQ LRS VCHCLYQVVS QRFPQNSIGA VGSAMFLRFI NPAIVSPYEA GILDKKPPPR IERGLKLMSK ILQSIANHVL FTKEEHM RP FNDFVKSNFD AARRFFLDIA SDCPTSDAVN HSLSFISDGN VLALHRLLWN NQEKIGQYLS SNRDHKAVGR RPFDKMAT L LAYLGPPEHK PVADTHWSSL NLTSSKFEEF MTRHQVHEKE EFKALKTLSI FYQAGTSKAG NPIFYYVARR FKTGQINGD LLIYHVLLTL KPYYAKPYEI VVDLTHTGPS NRFKTDFLSK WFVVFPGFAY DNVSAVYIYN CNSWVREYTK YHERLLTGLK GSKRLVFID CPGKLAEHIE HEQQKLPAAT LALEEDLKVF HNALKLAHKD TKVSIKVGST AVQVTSAERT KVLGQSVFLN D IYYASEIE EICLVDENQF TLTIANQGTP LTFMHQECEA IVQSIIHIRT RWELSQPDSI PQHTKIRPKD VPGTLLNIAL LN LGSSDPS LRSAAYNLLC ALTCTFNLKI EGQLLETSGL CIPANNTLFI VSISKTLAAN EPHLTLEFLE ECISGFSKSS IEL KHLCLE YMTPWLSNLV RFCKHNDDAK RQRVTAILDK LITMTINEKQ MYPSIQAKIW GSLGQITDLL DVVLDSFIKT SATG GLGSI KAEVMADTAV ALASGNVKLV SSKVIGRMCK IIDKTCLSPT PTLEQHLMWD DIAILARYML MLSFNNSLDV AAHLP YLFH VVTFLVATGP LSLRASTHGL VINIIHSLCT CSQLHFSEET KQVLRLSLTE FSLPKFYLLF GISKVKSAAV IAFRSS YRD RSFSPGSYER ETFALTSLET VTEALLEIME ACMRDIPTCK WLDQWTELAQ RFAFQYNPSL QPRALVVFGC ISKRVSH GQ IKQIIRILSK ALESCLKGPD TYNSQVLIEA TVIALTKLQP LLNKDSPLHK ALFWVAVAVL QLDEVNLYSA GTALLEQN L HTLDSLRIFN DKSPEEVFMA IRNPLEWHCK QMDHFVGLNF NSNFNFALVG HLLKGYRHPS PAIVARTVRI LHTLLTLVN KHRNCDKFEV NTQSVAYLAA LLTVSEEVRS RCSLKHRKSL LLTDISMENV PMDTYPIHHG DPSYRTLKET QPWSSPKGSE GYLAATYPT VGQTSPRARK SMSLDMGQPS QANTKKLLGT RKSFDHLISD TKAPKRQEME SGITTPPKMR RVAETDYEME T QRISSSQQ HPHLRKVSVS ESNVLLDEEV LTDPKIQALL LTVLATLVKY TTDEFDQRIL YEYLAEASVV FPKVFPVVHN LL DSKINTL LSLCQDPNLL NPIHGIVQSV VYHEESPPQY QTSYLQSFGF NGLWRFAGPF SKQTQIPDYA ELIVKFLDAL IDT YLPGID EETSEESLLT PTSPYPPALQ SQLSITANLN LSNSMTSLAT SQHSPGIDKE NVELSPTTGH CNSGRTRHGS ASQV QKQRS AGSFKRNSIK KIV

UniProtKB: Neurofibromin

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Macromolecule #2: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: GSP
Molecular weightTheoretical: 539.246 Da
Chemical component information

ChemComp-GSP:
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
20.0 mMC8H18N2O4SHEPES
10.0 mMC10H16N5O13P3SGTPgammaS
10.0 mMMgCl2Magnesium Chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN
Specialist opticsEnergy filter - Name: GIF 200
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 300000
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
PDB IDChain

6v65

chain_id: B, source_name: PDB, initial_model_type: experimental model

2e2x

chain_id: A, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7r04:
Neurofibromin in open conformation

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