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- PDB-9kax: The outward-open structure of BjSemiSWEET in native cellular memb... -

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Basic information

Entry
Database: PDB / ID: 9kax
TitleThe outward-open structure of BjSemiSWEET in native cellular membranes determined by in situ solid-state NMR
ComponentsSugar transporter SemiSWEET
KeywordsMEMBRANE PROTEIN / Membrane transporter / SemiSWEET / Outward-open / in situ Structure
Function / homology: / sugar transmembrane transporter activity / PQ-loop repeat / PQ loop repeat / plasma membrane / Sugar transporter SemiSWEET
Function and homology information
Biological speciesBradyrhizobium diazoefficiens USDA 110 (bacteria)
MethodSOLID-STATE NMR / simulated annealing
AuthorsXie, H. / Yang, J.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)22434003 China
National Natural Science Foundation of China (NSFC)22004124 China
Chinese Academy of SciencesYJKYYQ20190032 China
Chinese Academy of SciencesXDB0540000 China
CitationJournal: To Be Published
Title: Two functional conformations of a membrane transporter and their transition in native cellular membranes: distinct from those in synthetic membranes
Authors: Xie, H. / Yang, J.
History
DepositionOct 30, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sugar transporter SemiSWEET
B: Sugar transporter SemiSWEET


Theoretical massNumber of molelcules
Total (without water)18,3962
Polymers18,3962
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 1000structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Sugar transporter SemiSWEET


Mass: 9198.216 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bradyrhizobium diazoefficiens USDA 110 (bacteria)
Strain: Bradyrhizobium diazoefficiens USDA 110 (strain: USDA110
Gene: bsr6460 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli / References: UniProt: Q89G85
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D NCACX
121isotropic13D NCOCX
131isotropic13D CONCA
151isotropic12D 500ms CORD

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Sample preparation

DetailsType: membrane
Contents: 60 % w/w [U-13C; U-15N] Sucrose transport protein, Bradyrhizobium japonicum SemiSWEET (BjSemiSWEET), H2O
Details: BjSemiSWEET cellular membrane samples were derived from Escherichia coli cells. The total cellular membrane fraction was obtained through density gradient centrifugation and subsequently ...Details: BjSemiSWEET cellular membrane samples were derived from Escherichia coli cells. The total cellular membrane fraction was obtained through density gradient centrifugation and subsequently purified using an enhanced sucrose gradient centrifugation method50. The cellular membranes were resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, pH 8.2) and homogenized via gentle sonication before centrifugation. A layering technique was then applied, sequentially adding 2.7 mL of 60% (w/v) sucrose, 3.5 mL of 51% (w/v) sucrose, 3 mL of 35% (w/v) sucrose, and 4 mL of the total membrane suspension from bottom to top in a tube. Centrifugation was carried out using an SW-40Ti rotor (Beckman) at 169818 g for 16 hours. The inner membrane fraction between the 35% and 51% (w/v) sucrose layers was collected, as well as the outer membranes at the 51%-60% sucrose interface. To collect the inner membranes, 2 mL of the sample was diluted with water to fill an 8.9 mL Beckman centrifuge tube and centrifuged at 419832 g for 3 hours. After freeze-drying, rehydration with 30% (w/w) water was followed by packing into a 3.2 mm thin-walled rotor for solid-state NMR (ssNMR) experiments.
Label: [U-13C; U-15N] / Solvent system: H2O
SampleConc.: 60 % w/w
Component: Sucrose transport protein, Bradyrhizobium japonicum SemiSWEET (BjSemiSWEET)
Isotopic labeling: [U-13C; U-15N]
Sample conditionsDetails: BjSemiSWEET cellular membrane samples were derived from Escherichia coli cells. The total cellular membrane fraction was obtained through density gradient centrifugation and subsequently ...Details: BjSemiSWEET cellular membrane samples were derived from Escherichia coli cells. The total cellular membrane fraction was obtained through density gradient centrifugation and subsequently purified using an enhanced sucrose gradient centrifugation method50. The cellular membranes were resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, pH 8.2) and homogenized via gentle sonication before centrifugation. A layering technique was then applied, sequentially adding 2.7 mL of 60% (w/v) sucrose, 3.5 mL of 51% (w/v) sucrose, 3 mL of 35% (w/v) sucrose, and 4 mL of the total membrane suspension from bottom to top in a tube. Centrifugation was carried out using an SW-40Ti rotor (Beckman) at 169818 g for 16 hours. The inner membrane fraction between the 35% and 51% (w/v) sucrose layers was collected, as well as the outer membranes at the 51%-60% sucrose interface. To collect the inner membranes, 2 mL of the sample was diluted with water to fill an 8.9 mL Beckman centrifuge tube and centrifuged at 419832 g for 3 hours. After freeze-drying, rehydration with 30% (w/w) water was followed by packing into a 3.2 mm thin-walled rotor for solid-state NMR (ssNMR) experiments.
Ionic strength: 1 M / Ionic strength err: 0.2 / Label: [U-13C; U-15N] / pH: 8.2 / PH err: 0.1 / Pressure: 1 mbar / Pressure err: 0.01 / Temperature: 298 K / Temperature err: 0.2

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NMR measurement

NMR spectrometerType: Bruker Bruker AVANCE III 800 / Manufacturer: Bruker / Model: Bruker AVANCE III 800 / Field strength: 800 MHz
Details: Solid-state NMR experiments, including NCACX, NCOCX, CONCA, and NCACB, were conducted on BjSemiSWEET's cellular membrane samples using a standard Bruker Advance 800 MHz spectrometer equipped ...Details: Solid-state NMR experiments, including NCACX, NCOCX, CONCA, and NCACB, were conducted on BjSemiSWEET's cellular membrane samples using a standard Bruker Advance 800 MHz spectrometer equipped with a 3.2-mm E-free HCN MAS probe spinning at 10.5 kHz and 273 K.

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Processing

NMR software
NameDeveloperClassification
SparkyGoddardchemical shift assignment
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddardpeak picking
RefinementMethod: simulated annealing / Software ordinal: 2
Details: A total of 1000 structures were de novo generated through molecular dynamics simulated annealing in torsion angle space with two consecutive annealing schedules, followed by final gradient ...Details: A total of 1000 structures were de novo generated through molecular dynamics simulated annealing in torsion angle space with two consecutive annealing schedules, followed by final gradient minimization in Cartesian space. Each structure was subject to specific constraints: 379*2 unique non-redundant 13C-13C distance constraints, 74*2 hydrogen bond constraints, and 81*2 TALOS-N derived torsion angle constrained chains for comformation I, per BjSemiSWEET dimer structure.
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 1000 / Conformers submitted total number: 10

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