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- PDB-8t60: CryoEM structure of an inward-facing MelBSt at a Na(+)-bound and ... -
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Basic information
Entry | Database: PDB / ID: 8t60 | ||||||
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Title | CryoEM structure of an inward-facing MelBSt at a Na(+)-bound and sugar low-affinity conformation | ||||||
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![]() | TRANSPORT PROTEIN / Sugar transporter / Cation-coupled symporter / Na(+) binding / Protein conformation / Nanobodies / NabFab / CryoEM / Membrane proteins / protein-protein interaction | ||||||
Function / homology | ![]() : / symporter activity / sodium ion transport / carbohydrate transport / plasma membrane Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å | ||||||
![]() | Guan, L. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Mobile barrier mechanisms for Na-coupled symport in an MFS sugar transporter. Authors: Parameswaran Hariharan / Yuqi Shi / Satoshi Katsube / Katleen Willibal / Nathan D Burrows / Patrick Mitchell / Amirhossein Bakhtiiari / Samantha Stanfield / Els Pardon / H Ronald Kaback / ...Authors: Parameswaran Hariharan / Yuqi Shi / Satoshi Katsube / Katleen Willibal / Nathan D Burrows / Patrick Mitchell / Amirhossein Bakhtiiari / Samantha Stanfield / Els Pardon / H Ronald Kaback / Ruibin Liang / Jan Steyaert / Rosa Viner / Lan Guan / ![]() ![]() Abstract: While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial ...While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport. #1: ![]() Title: Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter Authors: Hariharan, P. / Shi, Y. / Katsube, S. / Willibal, K. / Burrows, N.D. / Mitchell, P. / Bakhtiiari, A. / Stanfield, S. / Pardon, E. / Kaback, H.R. / Liang, R. / Steyaert, J. / Viner, R. / Gran, L. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 427.3 KB | Display | ![]() |
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PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 41062MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 54104.438 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: LT2 / Gene: melB, STM4299 / Production host: ![]() ![]() |
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#2: Antibody | Mass: 14817.506 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: ![]() ![]() |
#3: Antibody | Mass: 25684.463 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: ![]() ![]() |
#4: Antibody | Mass: 23258.783 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: ![]() ![]() |
#5: Chemical | ChemComp-NA / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: MelBSt bound with Nb725_4-NabFab complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
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Molecular weight | Value: 0.12 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.0535 sec. / Electron dose: 1.28 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of real images: 20778 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
EM diffraction shell | Resolution: 3.29→5.5 Å / Fourier space coverage: 93.2 % / Multiplicity: 2.5 / Num. of structure factors: 244 / Phase residual: 13.5 ° |
EM diffraction stats | Fourier space coverage: 99 % / High resolution: 3.29 Å / Num. of intensities measured: 10000 / Num. of structure factors: 325 / Phase error rejection criteria: 0 / Rmerge: 10 |
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Processing
EM software |
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CTF correction | Details: using cryoSPARC patch CTF estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296925 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1
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Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 97.35 Å2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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