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- PDB-6r9t: Cryo-EM structure of autoinhibited human talin-1 -

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Basic information

Entry
Database: PDB / ID: 6r9t
TitleCryo-EM structure of autoinhibited human talin-1
ComponentsTalin-1
KeywordsCELL ADHESION / focal adhesion / signaling / actin / vinculin
Function / homology
Function and homology information


LIM domain binding / vinculin binding / XBP1(S) activates chaperone genes / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / integrin activation / cell-substrate junction assembly / cell-cell junction assembly / cortical actin cytoskeleton organization / regulation of focal adhesion assembly / phosphatidylserine binding ...LIM domain binding / vinculin binding / XBP1(S) activates chaperone genes / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / integrin activation / cell-substrate junction assembly / cell-cell junction assembly / cortical actin cytoskeleton organization / regulation of focal adhesion assembly / phosphatidylserine binding / p130Cas linkage to MAPK signaling for integrins / GRB2:SOS provides linkage to MAPK signaling for Integrins / Smooth Muscle Contraction / ruffle / phosphatidylinositol binding / Integrin signaling / integrin-mediated signaling pathway / adherens junction / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / structural constituent of cytoskeleton / cell-cell adhesion / platelet aggregation / ruffle membrane / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / actin filament binding / Signaling by BRAF and RAF1 fusions / integrin binding / Platelet degranulation / cytoskeleton / cadherin binding / focal adhesion / cell surface / extracellular exosome / extracellular region / plasma membrane / cytosol
Similarity search - Function
: / Talin, R4 domain / Vinculin-binding site-containing domain / Talin, central / Talin, N-terminal F0 domain / Talin, central domain superfamily / Talin-1/2, rod-segment / Vinculin Binding Site / Talin, middle domain / N-terminal or F0 domain of Talin-head FERM ...: / Talin, R4 domain / Vinculin-binding site-containing domain / Talin, central / Talin, N-terminal F0 domain / Talin, central domain superfamily / Talin-1/2, rod-segment / Vinculin Binding Site / Talin, middle domain / N-terminal or F0 domain of Talin-head FERM / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain / I/LWEQ domain profile. / I/LWEQ domain / Phosphotyrosine-binding domain / Alpha-catenin/vinculin-like superfamily / FERM domain signature 1. / FERM conserved site / FERM domain signature 2. / FERM central domain / FERM/acyl-CoA-binding protein superfamily / FERM central domain / FERM superfamily, second domain / FERM domain / FERM domain profile. / Band 4.1 domain / Band 4.1 homologues / PH-like domain superfamily / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsDedden, D. / Schumacher, S. / Zacharias, M. / Biertumpfel, C. / Mizuno, N.
Funding support Germany, 2items
OrganizationGrant numberCountry
European Research CouncilERC-CoG, 724209 Germany
Other privateBoehringer Ingelheim Stiftung Plus3 Germany
CitationJournal: Cell / Year: 2019
Title: The Architecture of Talin1 Reveals an Autoinhibition Mechanism.
Authors: Dirk Dedden / Stephanie Schumacher / Charlotte F Kelley / Martin Zacharias / Christian Biertümpfel / Reinhard Fässler / Naoko Mizuno /
Abstract: Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting ...Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.
History
DepositionApr 4, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.2Jul 29, 2020Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.phase_plate

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Structure visualization

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Assembly

Deposited unit
A: Talin-1


Theoretical massNumber of molelcules
Total (without water)270,7741
Polymers270,7741
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, monomer, light scattering, monomer, mass spectrometry, monomer, equilibrium centrifugation, monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area101910 Å2

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Components

#1: Protein Talin-1


Mass: 270773.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TLN1, KIAA1027, TLN / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold / References: UniProt: Q9Y490

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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: Talin-1 / Type: COMPLEX / Details: autoinhibited / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
275 mMKClKCl1
30.5 mMEDTAEthylenediaminetetraacetic acid(HO2CCH2)2NCH2CH2N(CH2CO2H)21
40.5 mMb-mercaptoethanol2-MercaptoethanolHSCH2CH2OH1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: GraFix
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K / Details: Blotted 4 seconds before plunging, blot force 4

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: May 14, 2018
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.62 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 76.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11007
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 4096 / Height: 4096 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4Gctf1.06CTF correction
7Coot0.8.9.1model fitting
8UCSF Chimera1.13.1model fitting
10cryoSPARC1initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIX1.14-3260model refinement
15Amber18model fitting
16Gautomatch0.56particle selection
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1873975
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30438 / Actual pixel size: 1.06 Å / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 377 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13IVF

3ivf

a1210-398
21SJ8

1sj8

a1486-785
32L7A

2l7a

a1786-910
42LQG

2lqg

a1911-1046
52L7N

2l7n

a11047-1205
62L10

2l10

a11206-1355
75IC1

5ic1

a11356-1822
82KVP

2kvp

a11823-1971
93DYJ

3dyj

a11972-2297
102JSW

2jsw

a12298-2479

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