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- PDB-9b3k: NorA in complex with Fab36 (NorA-BRIL fusion) -

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Basic information

Entry
Database: PDB / ID: 9b3k
TitleNorA in complex with Fab36 (NorA-BRIL fusion)
Components
  • Fab36 heavy chain
  • Fab36 light chain
  • NorA-BRIL(3A) fusion
KeywordsTRANSPORT PROTEIN/IMMUNE SYSTEM / membrane protein / Staphylococcus aureus / antibiotic resistance / efflux pump / TRANSPORT PROTEIN / TRANSPORT PROTEIN-IMMUNE SYSTEM complex
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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.56 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

Downloads & links

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Assembly

Deposited unit
Z: NorA-BRIL(3A) fusion
H: Fab36 heavy chain
L: Fab36 light chain


Theoretical massNumber of molelcules
Total (without water)107,2283
Polymers107,2283
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
#2: Antibody Fab36 heavy chain


Mass: 28044.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab36 light chain


Mass: 25794.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
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: NorA-BRIL(3A)-Fab36-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: MULTIPLE SOURCES
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 400 mM NaCl, 20 mM Tris
Buffer component
IDConc.NameFormulaBuffer-ID
1400 mMSodium ChlorideNaCl1
220 mMtris(hydroxymethyl)aminomethane1
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 to select particle images
4cryoSPARCv3.3.1CTF correctionPatch CTF estimation (multi) was used for CTF correction
7PHENIX1.20.1model fitting
9PHENIX1.20.1model refinement
10cryoSPARCv3.3.1initial Euler assignment
11cryoSPARCv3.3.1final Euler assignment
12cryoSPARCv3.3.1classification
13cryoSPARCv3.3.13D reconstruction
CTF correctionDetails: Patch CTF estimation (multi) was used for CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4031695 / Details: Topaz Training
3D reconstructionResolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298088 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7LO8

7lo8


Accession code: 7LO8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046207
ELECTRON MICROSCOPYf_angle_d0.5768421
ELECTRON MICROSCOPYf_dihedral_angle_d4.353844
ELECTRON MICROSCOPYf_chiral_restr0.043967
ELECTRON MICROSCOPYf_plane_restr0.0041045

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