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- EMDB-44145: NorA in inward-open conformation (NorA-BRIL fusion) -

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Basic information

Entry
Database: EMDB / ID: EMD-44145
TitleNorA in inward-open conformation (NorA-BRIL fusion)
Map data
Sample
  • Complex: NorA-BRIL(3A)-FabBRIL
    • Protein or peptide: NorA-BRIL(3A) fusion
Keywordsmembrane protein / Staphylococcus aureus / antibiotic resistance / efflux pump / TRANSPORT PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsXie P / Li Y / Kuang H / Wang DN / Traaseth NJ
Funding support United States, 5 items
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, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateJun 25, 2025-
Current statusJun 25, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44145.png

Downloads & links

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Map

FileDownload / File: emd_44145.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0902
Minimum - Maximum-1.7279979 - 2.5843928
Average (Standard dev.)0.00005060106 (±0.0375483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44145_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44145_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44145_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : NorA-BRIL(3A)-FabBRIL

EntireName: NorA-BRIL(3A)-FabBRIL
Components
  • Complex: NorA-BRIL(3A)-FabBRIL
    • Protein or peptide: NorA-BRIL(3A) fusion

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Supramolecule #1: NorA-BRIL(3A)-FabBRIL

SupramoleculeName: NorA-BRIL(3A)-FabBRIL / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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
Source (natural)Organism: Staphylococcus aureus (bacteria)

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

MacromoleculeName: NorA-BRIL(3A) fusion / type: protein_or_peptide / ID: 1
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)
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 53.388516 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL ...String:
MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL AFIMSIVLIH DPKKSTTSGF QKLEPQLLTK INWKVFITPV ILTLVLSFGL SAFETLYSLY TADKVNYSPK DI SIAITGG GIFGALFQIY FFDKFMKYFS ELTFIAWSLL YSVVVLILLV FANDYWSIML ISFVVFIGFD MIRPAITNYF SNI AGERQG FAGGLNSTFT SMGNFIGPLI AGALFDVHIE APIYMAIGVS LAGVVIVLIE KQHRAAAADL EDNWETLNDN LKVI EKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HGFDILVGQI DDALKLANEG KVKEAQAAAE QLKTT RNAY IQKYL

UniProtKB: Quinolone resistance protein NorA

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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

BufferpH: 5
Component:
ConcentrationFormulaName
400.0 mMNaClSodium Chloride
50.0 mMSodium Acetate

Details: 400 mM NaCl, 50 mM sodium acetate, pH 5.0
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER / Details: hold for 10 sec before 25sec glow discharging
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 1.2 sec. / Average electron dose: 55.54 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: 1.0 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 1812072 / Details: Topaz Training
CTF correctionSoftware - Name: cryoSPARC (ver. v3.3.1) / Software - details: Patch CTF estimation
Details: Patch CTF estimation (multi) was used for CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab0initio reconstruction
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 159621
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v3.3.1) / Details: Branch-and-bound Maximum Likelihood
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v3.3.1) / Details: Branch-and-bound Maximum Likelihood
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. v3.3.1)
Details: 2 classes contain 53756 and 105865 particles respectively
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: other / Details: Alphafold2 prediction
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9b3m:
NorA in inward-open conformation (NorA-BRIL fusion)

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