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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 data
SampleComplex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
  • Multidrug resistance protein NorMMultiple drug resistance
  • NabFab HC
  • NabFab LC
  • NorM-Nb17_4
  • Anti-Fab nanobody
Function / homologyMulti antimicrobial extrusion protein / MatE / antiporter activity / xenobiotic transmembrane transporter activity / integral component of membrane / 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 450 pix.
= 301.5 Å
0.67 Å/pix.
x 450 pix.
= 301.5 Å
0.67 Å/pix.
x 450 pix.
= 301.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Number of Components: 6

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Component #1: protein, Complex of vibrio cholerae MATE transporter NorM, chimer...

ProteinName: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
Recombinant expression: No
SourceSpecies: Vibrio cholerae RC385 (bacteria)

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Component #2: protein, Multidrug resistance protein NorM

ProteinName: Multidrug resistance protein NorMMultiple drug resistance
Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, NabFab HC

ProteinName: NabFab HC / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, NabFab LC

ProteinName: NabFab LC / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, NorM-Nb17_4

ProteinName: NorM-Nb17_4 / Recombinant expression: No
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Anti-Fab nanobody

ProteinName: Anti-Fab nanobody / Recombinant expression: No

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen Name: OTHER

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 26 e/Å2 / Illumination Mode: SPOT SCAN
LensImaging Mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Number of Projections: 433559
3D reconstructionResolution: 3.68 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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