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- PDB-7php: Structure of Multidrug and Toxin Compound Extrusion (MATE) transp... -

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Entry
Database: PDB / ID: 7php
TitleStructure of Multidrug and Toxin Compound Extrusion (MATE) transporter NorM by NabFab-fiducial assisted cryo-EM
Components
  • Anti-Fab nanobody
  • Multidrug resistance protein NorMMultiple drug resistance
  • NabFab HC
  • NabFab LC
  • NorM-Nb17_4
KeywordsMEMBRANE PROTEIN / NabFab / anti-nanobody Fab / fiducial / NorM / MATE
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsBloch, J.S. / Mukherjee, S. / Kowal, J. / Niederer, M. / Pardon, E. / Steyaert, J. / Kossiakoff, A.A. / Locher, K.P.
Funding support Switzerland, 1items
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, J.S. / Mukherjee, S. / Kowal, J. / Filippova, E.V. / Niederer, M. / Pardon, E. / Steyaert, J. / Kossiakoff, A.A. / Locher, K.P.
History
DepositionAug 18, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Source and taxonomy
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / em_admin / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / pdbx_database_proc
Item: _em_admin.last_update
Revision 1.2Dec 1, 2021Group: Database references / Category: citation / citation_author

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Multidrug resistance protein NorM
H: NabFab HC
L: NabFab LC
N: NorM-Nb17_4
K: Anti-Fab nanobody


Theoretical massNumber of molelcules
Total (without water)129,1535
Polymers129,1535
Non-polymers00
Water37821
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8190 Å2
ΔGint-45 kcal/mol
Surface area51090 Å2

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Components

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Antibody , 4 types, 4 molecules HLNK

#2: Antibody NabFab HC


Mass: 25684.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody NabFab LC


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody NorM-Nb17_4


Mass: 14356.979 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Antibody Anti-Fab nanobody


Mass: 13390.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Protein / Non-polymers , 2 types, 22 molecules A

#1: Protein Multidrug resistance protein NorM / Multiple drug resistance / Na(+)/drug antiporter


Mass: 52462.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae RC385 (bacteria) / Gene: VCRC385_01100 / Production host: Escherichia coli (E. coli) / References: UniProt: D7H904
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O

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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: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Vibrio cholerae RC385 (bacteria)345074
21synthetic construct (others)32630
31synthetic construct (others)32630
41synthetic construct (others)32630
51Lama glama (llama)9844
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 26 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 433995 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0068819
ELECTRON MICROSCOPYf_angle_d0.68911985
ELECTRON MICROSCOPYf_dihedral_angle_d8.7321222
ELECTRON MICROSCOPYf_chiral_restr0.0461375
ELECTRON MICROSCOPYf_plane_restr0.0051503

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