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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
TitleCryoEM structure of an inward-facing MelBSt at a Na(+)-bound and sugar low-affinity conformation
Components
  • Melibiose permease
  • NabFab_H Chain
  • NabFab_L Chain
  • Nb725_4
KeywordsTRANSPORT PROTEIN / Sugar transporter / Cation-coupled symporter / Na(+) binding / Protein conformation / Nanobodies / NabFab / CryoEM / Membrane proteins / protein-protein interaction
Function / homology
Function and homology information


symporter activity / sodium ion transport / carbohydrate transport / transmembrane transport / plasma membrane
Similarity search - Function
Sodium:galactoside symporter / Sodium:galactoside symporter, conserved site / Sodium:galactoside symporter family signature. / Lactose permease-like / MFS/sugar transport protein / MFS transporter superfamily
Similarity search - Domain/homology
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsGuan, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122759 United States
Citation
Journal: Elife / Year: 2024
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: Journal: Elife / Year: 2024
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
DepositionJun 14, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author
Revision 1.2Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Melibiose permease
B: Nb725_4
H: NabFab_H Chain
L: NabFab_L Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,8885
Polymers117,8654
Non-polymers231
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Melibiose permease / Melibiose carrier / Melibiose transporter / Melibiose/cation symporter / Na+ (Li+)/melibiose ...Melibiose carrier / Melibiose transporter / Melibiose/cation symporter / Na+ (Li+)/melibiose symporter / Thiomethylgalactoside permease II


Mass: 54104.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: LT2 / Gene: melB, STM4299 / Production host: Escherichia coli (E. coli) / References: UniProt: P30878
#2: Antibody Nb725_4


Mass: 14817.506 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody NabFab_H Chain


Mass: 25684.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody NabFab_L Chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: MelBSt bound with Nb725_4-NabFab complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE

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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: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum
EM diffraction shellResolution: 3.29→5.5 Å / Fourier space coverage: 93.2 % / Multiplicity: 2.5 / Num. of structure factors: 244 / Phase residual: 13.5 °
EM diffraction statsFourier 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
IDNameVersionCategory
1cryoSPARCv3particle selection
2EPUimage acquisition
4cryoSPARCv3CTF correction
7UCSF ChimeraXmodel fitting
8PHENIXmodel fitting
10PHENIX1.2model refinement
13cryoSPARCv4classification
14cryoSPARCcryoSPARC3D reconstruction
CTF correctionDetails: using cryoSPARC patch CTF estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296925 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue rangeSource nameType
17L17

7l17

A7L17A2-45412-454PDBexperimental model
2AlphaFoldin silico model
37PHP

7php

H7PHPH2-21432-214PDBexperimental model
47PHP

7php

L7PHPL4-21134-211PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 97.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00287690
ELECTRON MICROSCOPYf_angle_d0.497810474
ELECTRON MICROSCOPYf_chiral_restr0.03981199
ELECTRON MICROSCOPYf_plane_restr0.00281303
ELECTRON MICROSCOPYf_dihedral_angle_d11.07012655

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