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- EMDB-13425: Map of complex of Vibrio cholerae MATE transporter NorM, chimeric... -

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Basic information

Entry
Database: EMDB / ID: EMD-13425
TitleMap of complex of Vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody with fulcrum in center of micelle
Map dataMap of VcNorM NabFab complex with fulcrum in center of micelle
Sample
  • Complex: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
    • Complex: Multidrug resistance protein NorMMultiple drug resistance
    • Complex: NabFab HC
    • Complex: NabFab LC
    • Complex: NorM-Nb17_4
    • Complex: Anti-Fab nanobody
Function / homologyMulti antimicrobial extrusion protein / MatE / antiporter activity / xenobiotic transmembrane transporter activity / membrane => GO:0016020 / Multidrug resistance protein NorM
Function and homology information
Biological speciesVibrio cholerae RC385 (bacteria) / synthetic construct (others) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsBloch JS / Mukherjee S / Kowal J / Niederer M / Pardon E / Steyaert J / Kossiakoff AA / Locher KP
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2021
Title: Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
Authors: Joël S Bloch / Somnath Mukherjee / Julia Kowal / Ekaterina V Filippova / Martina Niederer / Els Pardon / Jan Steyaert / Anthony A Kossiakoff / Kaspar P Locher /
Abstract: With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that ...With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in in large amounts, it may be useful not only for structural biology but also for biomedical applications.
#1: Journal: Biorxiv / Year: 2021
Title: Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins
Authors: Bloch JS / Mukherjee S / Kowal J / Filippova EV / Niederer M / Pardon E / Steyaert J / Kossiakoff AA / Locher KP
History
DepositionAug 18, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0129
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0129
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13425.png

Downloads & links

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Map

FileDownload / File: emd_13425.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of VcNorM NabFab complex with fulcrum in center of micelle
Voxel sizeX=Y=Z: 0.67 Å
Density
Contour LevelBy AUTHOR: 0.0129 / Movie #1: 0.0129
Minimum - Maximum-0.034917712 - 0.060584508
Average (Standard dev.)8.4917665e-06 (±0.0012790977)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 301.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.670.670.67
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z301.500301.500301.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS450450450
D min/max/mean-0.0350.0610.000

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Supplemental data

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Sample components

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Entire : Complex of vibrio cholerae MATE transporter NorM, chimeric nanobo...

EntireName: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
Components
  • Complex: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
    • Complex: Multidrug resistance protein NorMMultiple drug resistance
    • Complex: NabFab HC
    • Complex: NabFab LC
    • Complex: NorM-Nb17_4
    • Complex: Anti-Fab nanobody

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Supramolecule #1: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobo...

SupramoleculeName: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Vibrio cholerae RC385 (bacteria)

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Supramolecule #2: Multidrug resistance protein NorM

SupramoleculeName: Multidrug resistance protein NorM / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: NabFab HC

SupramoleculeName: NabFab HC / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: NabFab LC

SupramoleculeName: NabFab LC / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #5: NorM-Nb17_4

SupramoleculeName: NorM-Nb17_4 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #6: Anti-Fab nanobody

SupramoleculeName: Anti-Fab nanobody / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #5

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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: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 26.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 433559
FSC plot (resolution estimation)

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