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- EMDB-11817: Barbed tail of the human TSC protein complex -

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Entry
Database: EMDB / ID: EMD-11817
TitleBarbed tail of the human TSC protein complex
Map dataBarbed tail of the human TSC protein complex
Sample
  • Complex: Barbed tail of the human TSC protein complex
    • Protein or peptide: Hamartin
    • Protein or peptide: Tuberin
    • Protein or peptide: TBC1D7
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsRamlaul K / Fu W / Wu G / Aylett CHS
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust206212/Z/17/Z United Kingdom
CitationJournal: J Mol Biol / Year: 2021
Title: Architecture of the Tuberous Sclerosis Protein Complex.
Authors: Kailash Ramlaul / Wencheng Fu / Hua Li / Natàlia de Martin Garrido / Lin He / Manjari Trivedi / Wei Cui / Christopher H S Aylett / Geng Wu /
Abstract: The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream ...The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.
History
DepositionOct 5, 2020-
Header (metadata) releaseDec 30, 2020-
Map releaseDec 30, 2020-
UpdateJan 13, 2021-
Current statusJan 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11817.png

Downloads & links

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Map

FileDownload / File: emd_11817.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBarbed tail of the human TSC protein complex
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.004838834 - 0.020552367
Average (Standard dev.)3.047022e-08 (±0.00037139966)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 518.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z518.400518.400518.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ540540540
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0050.0210.000

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Supplemental data

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Half map: Half map 2

Fileemd_11817_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_11817_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Barbed tail of the human TSC protein complex

EntireName: Barbed tail of the human TSC protein complex
Components
  • Complex: Barbed tail of the human TSC protein complex
    • Protein or peptide: Hamartin
    • Protein or peptide: Tuberin
    • Protein or peptide: TBC1D7

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Supramolecule #1: Barbed tail of the human TSC protein complex

SupramoleculeName: Barbed tail of the human TSC protein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 700 KDa

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Macromolecule #1: Hamartin

MacromoleculeName: Hamartin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAQQANVGEL LAMLDSPMLG VRDDVTAVFK ENLNSDRGPM LVNTLVDYYL ETSSQPALHI LTTLQEPHD KHLLDRINEY VGKAATRLSI LSLLGHVIRL QPSWKHKLSQ APLLPSLLKC L KMDTDVVV LTTGVLVLIT MLPMIPQSGK QHLLDFFDIF GRLSSWCLKK ...String:
MAQQANVGEL LAMLDSPMLG VRDDVTAVFK ENLNSDRGPM LVNTLVDYYL ETSSQPALHI LTTLQEPHD KHLLDRINEY VGKAATRLSI LSLLGHVIRL QPSWKHKLSQ APLLPSLLKC L KMDTDVVV LTTGVLVLIT MLPMIPQSGK QHLLDFFDIF GRLSSWCLKK PGHVAEVYLV HL HASVYAL FHRLYGMYPC NFVSFLRSHY SMKENLETFE EVVKPMMEHV RIHPELVTGS KDH ELDPRR WKRLETHDVV IECAKISLDP TEASYEDGYS VSHQISARFP HRSADVTTSP YADT QNSYG CATSTPYSTS RLMLLNMPGQ LPQTLSSPST RLITEPPQAT LWSPSMVCGM TTPPT SPGN VPPDLSHPYS KVFGTTAGGK GTPLGTPATS PPPAPLCHSD DYVHISLPQA TVTPPR KEE RMDSARPCLH RQHHLLNDRG SEEPPGSKGS VTLSDLPGFL GDLASEEDSI EKDKEEA AI SRELSEITTA EAEPVVPRGG FDSPFYRDSL PGSQRKTHSA ASSSQGASVN PEPLHSSL D KLGPDTPKQA FTPIDLPCGS ADESPAGDRE CQTSLETSIF TPSPCKIPPP TRVGFGSGQ PPPYDHLFEV ALPKTAHHFV IRKTEELLKK AKGNTEEDGV PSTSPMEVLD RLIQQGADAH SKELNKLPL PSKSVDWTHF GGSPPSDEIR TLRDQLLLLH NQLLYERFKR QQHALRNRRL L RKVIKAAA LEEHNAAMKD QLKLQEKDIQ MWKVSLQKEQ ARYNQLQEQR DTMVTKLHSQ IR QLQHDRE EFYNQSQELQ TKLEDCRNMI AELRIELKKA NNKVCHTELL LSQVSQKLSN SES VQQQME FLNRQLLVLG EVNELYLEQL QNKHSDTTKE VEMMKAAYRK ELEKNRSHVL QQTQ RLDTS QKRILELESH LAKKDHLLLE QKKYLEDVKL QARGQLQAAE SRYEAQKRIT QVFEL EILD LYGRLEKDGL LKKLEEEKAE AAEAAEERLD CCNDGCSDSM VGHNEEASGH NGETKT PRP SSARGSSGSR GGGGSSSSSS ELSTPEKPPH QRAGPFSSRW ETTMGEASAS IPTTVGS LP SSKSFLGMKA RELFRNKSES QCDEDGMTSS LSESLKTELG KDLGVEAKIP LNLDGPHP S PPTPDSVGQL HIMDYNETHH EHS

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Macromolecule #2: Tuberin

MacromoleculeName: Tuberin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAKPTSKDSG LKEKFKILLG LGTPRPNPRS AEGKQTEFII TAEILRELSM ECGLNNRIRM IGQICEVAK TKKFEEHAVE ALWKAVADLL QPERPLEARH AVLALLKAIV QGQGERLGVL R ALFFKVIK DYPSNEDLHE RLEVFKALTD NGRHITYLEE ELADFVLQWM ...String:
MAKPTSKDSG LKEKFKILLG LGTPRPNPRS AEGKQTEFII TAEILRELSM ECGLNNRIRM IGQICEVAK TKKFEEHAVE ALWKAVADLL QPERPLEARH AVLALLKAIV QGQGERLGVL R ALFFKVIK DYPSNEDLHE RLEVFKALTD NGRHITYLEE ELADFVLQWM DVGLSSEFLL VL VNLVKFN SCYLDEYIAR MVQMICLLCV RTASSVDIEV SLQVLDAVVC YNCLPAESLP LFI VTLCRT INVKELCEPC WKLMRNLLGT HLGHSAIYNM CHLMEDRAYM EDAPLLRGAV FFVG MALWG AHRLYSLRNS PTSVLPSFYQ AMACPNEVVS YEIVLSITRL IKKYRKELQV VAWDI LLNI IERLLQQLQT LDSPELRTIV HDLLTTVEEL CDQNEFHGSQ ERYFELVERC ADQRPE SSL LNLISYRAQS IHPAKDGWIQ NLQALMERFF RSESRGAVRI KVLDVLSFVL LINRQFY EE ELINSVVISQ LSHIPEDKDH QVRKLATQLL VDLAEGCHTH HFNSLLDIIE KVMARSLS P PPELEERDVA AYSASLEDVK TAVLGLLVIL QTKLYTLPAS HATRVYEMLV SHIQLHYKH SYTLPIASSI RLQAFDFLLL LRADSLHRLG LPNKDGVVRF SPYCVCDYME PERGSEKKTS GPLSPPTGP PGPAPAGPAV RLGSVPYSLL FRVLLQCLKQ ESDWKVLKLV LGRLPESLRY K VLIFTSPC SVDQLCSALC SMLSGPKTLE RLRGAPEGFS RTDLHLAVVP VLTALISYHN YL DKTKQRE MVYCLEQGLI HRCASQCVVA LSICSVEMPD IIIKALPVLV VKLTHISATA SMA VPLLEF LSTLARLPHL YRNFAAEQYA SVFAISLPYT NPSKFNQYIV CLAHHVIAMW FIRC RLPFR KDFVPFITKG LRSNVLLSFD DTPEKDSFRA RSTSLNERPK SLRIARPPKQ GLNNS PPVK EFKESSAAEA FRCRSISVSE HVVRSRIQTS LTSASLGSAD ENSVAQADDS LKNLHL ELT ETCLDMMARY VFSNFTAVPK RSPVGEFLLA GGRTKTWLVG NKLVTVTTSV GTGTRSL LG LDSGELQSGP ESSSSPGVHV RQTKEAPAKL ESQAGQQVSR GARDRVRSMS GGHGLRVG A LDVPASQFLG SATSPGPRTA PAAKPEKASA GTRVPVQEKT NLAAYVPLLT QGWAEILVR RPTGNTSWLM SLENPLSPFS SDINNMPLQE LSNALMAAER FKEHRDTALY KSLSVPAAST AKPPPLPRS NTVASFSSLY QSSCQGQLHR SVSWADSAVV MEEGSPGEVP VLVEPPGLED V EAALGMDR RTDAYSRSSS VSSQEEKSLH AEELVGRGIP IERVVSSEGG RPSVDLSFQP SQ PLSKSSS SPELQTLQDI LGDPGDKADV GRLSPEVKAR SQSGTLDGES AAWSASGEDS RGQ PEGPLP SSSPRSPSGL RPRGYTISDS APSRRGKRVE RDALKSRATA SNAEKVPGIN PSFV FLQLY HSPFFGDESN KPILLPNESQ SFERSVQLLD QIPSYDTHKI AVLYVGEGQS NSELA ILSN EHGSYRYTEF LTGLGRLIEL KDCQPDKVYL GGLDVCGEDG QFTYCWHDDI MQAVFH IAT LMPTKDVDKH RCDKKRHLGN DFVSIVYNDS GEDFKLGTIK GQFNFVHVIV TPLDYEC NL VSLQCRKDME GLVDTSVAKI VSDRNLPFVA RQMALHANMA SQVHHSRSNP TDIYPSKW I ARLRHIKRLR QRICEEAAYS NPSLPLVHPP SHSKAPAQTP AEPTPGYEVG QRKRLISSV EDFTEFV

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Macromolecule #3: TBC1D7

MacromoleculeName: TBC1D7 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MTEDSQRNFR SVYYEKVGFR GVEEKKSLEI LLKDDRLDTE KLCTFSQRFP LPSMYRALVW KVLLGILPP HHESHAKVMM YRKEQYLDVL HALKVVRFVS DATPQAEVYL RMYQLESGKL P RSPSFPLE PDDEVFLAIA KAMEEMVEDS VDCYWITRRF VNQLNTKYRD ...String:
MTEDSQRNFR SVYYEKVGFR GVEEKKSLEI LLKDDRLDTE KLCTFSQRFP LPSMYRALVW KVLLGILPP HHESHAKVMM YRKEQYLDVL HALKVVRFVS DATPQAEVYL RMYQLESGKL P RSPSFPLE PDDEVFLAIA KAMEEMVEDS VDCYWITRRF VNQLNTKYRD SLPQLPKAFE QY LNLEDGR LLTHLRMCSA APKLPYDLWF KRCFAGCLPE SSLQRVWDKV VSGSCKILVF VAV EILLTF KIKVMALNSA EKITKFLENI PQDSSDAIVS KAIDLWHKHC GTPVHSS

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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

Concentration0.2 mg/mL
BufferpH: 7.6
Details: 25 mM K-HEPES, pH 7.6, 175 mM KCl or 150 mM LiCl, 1 mM TCEP, and 0.5 uM EDTA
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY ARRAY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: 2s blot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 70 eV
DetailsData collected at eBIC
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 5387 / Average electron dose: 52.3 e/Å2 / Details: Dose weighting
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination 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: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAlignment and dose-weighting performed using motioncor2
Particle selectionDetails: Particles were selected in multiple rounds using BOXER and then TOPAZ before subregions were extracted based on vectors from initial 3D reconstructions
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Details: CTFs determined with CTFFIND 4, then filtering performed during reconstruction in RELION 3
Startup modelType of model: OTHER / Details: EMAN initial model from negative stain data
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Details: RELION 3 AUTOREFINE / Number images used: 58307
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 15.0 degrees
Software - Name: RELION (ver. 3) / Details: RELION 3 - HEALPIX 1
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 0.9375 degrees
Software - Name: RELION (ver. 3) / Details: RELION 3 - HEALPIX 5

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