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- EMDB-41062: CryoEM structure of an inward-facing MelBSt at a Na(+)-bound and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41062
TitleCryoEM structure of an inward-facing MelBSt at a Na(+)-bound and sugar low-affinity conformation
Map dataThe model was refined against this map.
Sample
  • Complex: MelBSt bound with Nb725_4-NabFab complex
    • Protein or peptide: Melibiose permease
    • Protein or peptide: Nb725_4
    • Protein or peptide: NabFab_H Chain
    • Protein or peptide: NabFab_L Chain
  • Ligand: SODIUM ION
KeywordsSugar transporter / Cation-coupled symporter / Na(+) binding / Protein conformation / Nanobodies / NabFab / CryoEM / Membrane proteins / protein-protein interaction / TRANSPORT PROTEIN
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 speciesEscherichia coli (E. coli) / Salmonella enterica subsp. enterica serovar Typhimurium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsGuan L
Funding support United States, 1 items
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 ND / Mitchell P / Bakhtiiari A / Stanfield S / Pardon E / Kaback HR / Liang R / Steyaert J / Viner R / Gran L
History
DepositionJun 14, 2023-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41062.png

Downloads & links

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Map

FileDownload / File: emd_41062.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe model was refined against this map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.7
Minimum - Maximum-31.29345 - 59.314101999999998
Average (Standard dev.)-0.000000000005194 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41062_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_41062_additional_1.map
Projections & Slices
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Half map: #2

Fileemd_41062_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_41062_half_map_2.map
Projections & Slices
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Sample components

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Entire : MelBSt bound with Nb725_4-NabFab complex

EntireName: MelBSt bound with Nb725_4-NabFab complex
Components
  • Complex: MelBSt bound with Nb725_4-NabFab complex
    • Protein or peptide: Melibiose permease
    • Protein or peptide: Nb725_4
    • Protein or peptide: NabFab_H Chain
    • Protein or peptide: NabFab_L Chain
  • Ligand: SODIUM ION

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Supramolecule #1: MelBSt bound with Nb725_4-NabFab complex

SupramoleculeName: MelBSt bound with Nb725_4-NabFab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Melibiose permease

MacromoleculeName: Melibiose permease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: LT2
Molecular weightTheoretical: 54.104438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SISMTTKLSY GFGAFGKDFA IGIVYMYLMY YYTDVVGLSV GLVGTLFLVA RIWDAINDPI MGWIVNATRS RWGKFKPWIL IGTLTNSLV LFLLFSAHLF EGTAQVVFVC VTYILWGMTY TIMDIPFWSL VPTITLDKRE REQLVPFPRF FASLAGFVTA G ITLPFVSY ...String:
SISMTTKLSY GFGAFGKDFA IGIVYMYLMY YYTDVVGLSV GLVGTLFLVA RIWDAINDPI MGWIVNATRS RWGKFKPWIL IGTLTNSLV LFLLFSAHLF EGTAQVVFVC VTYILWGMTY TIMDIPFWSL VPTITLDKRE REQLVPFPRF FASLAGFVTA G ITLPFVSY VGGADRGFGF QMFTLVLIAF FIASTIVTLR NVHEVYSSDN GVTAGRPHLT LKTIVGLIYK NDQLSCLLGM AL AYNIASN IINGFAIYYF TYVIGDADLF PYYLSYAGAA NLLTLIVFPR LVKMLSRRIL WAGASVMPVL SCAGLFAMAL ADI HNAALI VAAGIFLNIG TALFWVLQVI MVADTVDYGE FKLNIRCESI AYSVQTMVVK GGSAFAAFFI ALVLGLIGYT PNVA QSAQT LQGMQFIMIV LPVLFFMMTL VLYFRYYRLN GDMLRKIQIH LLDKYRKTPP FVEQPDSPAI SVVATSDVKA HHHHH HHHH H

UniProtKB: Melibiose permease

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Macromolecule #2: Nb725_4

MacromoleculeName: Nb725_4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.817506 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSQRQLVESG GGLVQPGGSL RLSCAVSGII FRDNAMGWYR QAPGKEREWV ATITDLGYTA YADSVKGRFT ISRDNAKDTV YLQMNSLEP EDTAVYYCHL PGTAAGDYWG KGTPVTVSSL EVLFQGPHHH HHHHH

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Macromolecule #3: NabFab_H Chain

MacromoleculeName: NabFab_H Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.684463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH T

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Macromolecule #4: NabFab_L Chain

MacromoleculeName: NabFab_L Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #5: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 5 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Number real images: 20778 / Average exposure time: 0.0535 sec. / Average electron dose: 1.28 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
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

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7l17


Details: MelBst, 7L17 NabFab, 7PHP Nb725_4, alphafold 2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. cryoSPARC) / Number images used: 296925
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7l17

chain_id: A, residue_range: 2-454, source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico model

7php

chain_id: H, residue_range: 2-214, source_name: PDB, initial_model_type: experimental model

7php

chain_id: L, residue_range: 4-211, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-8t60:
CryoEM structure of an inward-facing MelBSt at a Na(+)-bound and sugar low-affinity conformation

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