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- PDB-9b3o: NorA in inward-occluded conformation (NorA-BRIL fusion) -

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Basic information

Entry
Database: PDB / ID: 9b3o
TitleNorA in inward-occluded conformation (NorA-BRIL fusion)
ComponentsNorA-BRIL(3A) fusion
KeywordsTRANSPORT PROTEIN / membrane protein / Staphylococcus aureus / antibiotic resistance / efflux pump
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / plasma membrane
Similarity search - Function
Tetracycline resistance protein TetA/multidrug resistance protein MdtG / : / Multidrug resistance protein / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Quinolone resistance protein NorA
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsXie, P. / Li, Y. / Kuang, H. / Wang, D.N. / Traaseth, N.J.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI165782 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI108889 United States
National Science Foundation (NSF, United States)MCB1902449 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS108151 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK135088 United States
CitationJournal: Nat Commun / Year: 2025
Title: A fiducial-assisted strategy compatible with resolving small MFS transporter structures in multiple conformations using cryo-EM.
Authors: Pujun Xie / Yan Li / Gaëlle Lamon / Huihui Kuang / Da-Neng Wang / Nathaniel J Traaseth /
Abstract: Advancements in cryo-EM have stimulated a revolution in structural biology. Yet, for membrane proteins near the cryo-EM size threshold of approximately 40 kDa, including transporters and G-protein ...Advancements in cryo-EM have stimulated a revolution in structural biology. Yet, for membrane proteins near the cryo-EM size threshold of approximately 40 kDa, including transporters and G-protein coupled receptors, the absence of distinguishable structural features makes image alignment and structure determination a significant challenge. Furthermore, resolving more than one protein conformation within a sample, a major advantage of cryo-EM, represents an even greater degree of difficulty. Here, we describe a strategy for introducing a rigid fiducial marker (BRIL domain) at the C-terminus of membrane transporters from the Major Facilitator Superfamily (MFS) with AlphaFold2. This approach involves fusion of the last transmembrane domain helix of the target protein with the first helix of BRIL through a short poly-alanine linker to promote helicity. Combining this strategy with a BRIL-specific Fab, we elucidated four cryo-EM structures of the 42 kDa Staphylococcus aureus transporter NorA, three of which were derived from a single sample corresponding to inward-open, inward-occluded, and occluded conformations. Hence, this fusion construct facilitated experiments to characterize the conformational landscape of NorA and validated our design to position the BRIL/antibody pair in an orientation that avoids steric clash with the transporter. The latter was enabled through AlphaFold2 predictions, which minimized guesswork and reduced the need for screening several constructs. We further validated the suitability of the method to three additional MFS transporters (GlpT, Bmr, and Blt), results that supported a rigid linker between the transporter and BRIL. The successful application to four MFS proteins, the largest family of secondary transporters in nature, and analysis of predicted structures for the family indicates this strategy will be a valuable tool for studying other MFS members using cryo-EM.
History
DepositionMar 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NorA-BRIL(3A) fusion


Theoretical massNumber of molelcules
Total (without water)53,3891
Polymers53,3891
Non-polymers00
Water00
1


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

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Components

#1: Protein NorA-BRIL(3A) fusion


Mass: 53388.516 Da / Num. of mol.: 1
Fragment: NorA domain (1-380) + 3A linker (381-383) + Bril domain (384-490)
Source method: isolated from a genetically manipulated source
Details: NorA-BRIL(3A) fusion details: M1 to R380 is NorA domain; A381 to A383 is the 3A linker; A384 to L490 is Bril domain (most residues for Bril domain were not built since a mask was added only ...Details: NorA-BRIL(3A) fusion details: M1 to R380 is NorA domain; A381 to A383 is the 3A linker; A384 to L490 is Bril domain (most residues for Bril domain were not built since a mask was added only around NorA domain during data processing)
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: norA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q53459
Has protein modificationN

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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: NorA-BRIL(3A)-FabBRIL / Type: COMPLEX
Details: FabBRIL was added but not built in model due to masking. Here is its sequence: Heavy chain: ...Details: FabBRIL was added but not built in model due to masking. Here is its sequence: Heavy chain: SEVQLVESGGGLVQPGGSLRLSCAASGFNVVDFSLHWVRQAPGKGLEWVAYISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWGYWPGEPWWKAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS Light chain: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYLYYSLVTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 5 / Details: 400 mM NaCl, 50 mM sodium acetate, pH 5.0
Buffer component
IDConc.NameFormulaBuffer-ID
1400 mMSodium ChlorideNaCl1
250 mMSodium Acetate1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: hold for 10 sec before 25sec glow discharging / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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 magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 55.54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selectionTopaz training used
4cryoSPARCv3.3.1CTF correctionPatch CTF estimation (multi)
7PHENIX1.20.1model fitting
9cryoSPARCv3.3.1initial Euler assignment
10cryoSPARCv3.3.1final Euler assignment
11cryoSPARCv3.3.1classification
12cryoSPARCv3.3.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionDetails: Patch CTF estimation (multi) was used for CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1812072 / Details: Topaz training
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117232 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: Alphafold2 prediction / Source name: Other / Type: other
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032694
ELECTRON MICROSCOPYf_angle_d0.5873665
ELECTRON MICROSCOPYf_dihedral_angle_d3.844374
ELECTRON MICROSCOPYf_chiral_restr0.038440
ELECTRON MICROSCOPYf_plane_restr0.004448

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