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- PDB-7phq: Structure of homo-dimeric Staphylococcus capitis divalent metal i... -

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Basic information

Entry
Database: PDB / ID: 7phq
TitleStructure of homo-dimeric Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM
Components
  • Anti-Fab nanobody
  • DMT-Nb16_4
  • Divalent metal cation transporter MntH
  • NabFab HC
  • NabFab LC
KeywordsMEMBRANE PROTEIN / NabFab / Anti-nanobody Fab / Fiducial / DMT
Function / homologymetal ion transmembrane transporter activity / NRAMP family / Natural resistance-associated macrophage protein / symporter activity / integral component of membrane / plasma membrane / metal ion binding / Divalent metal cation transporter MntH
Function and homology information
Biological speciessynthetic construct (others)
Staphylococcus capitis (bacteria)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.45 Å
AuthorsBloch, J.S. / Mukherjee, S. / Kowal, 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 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
H: NabFab HC
L: NabFab LC
B: Divalent metal cation transporter MntH
N: DMT-Nb16_4
K: Anti-Fab nanobody
A: NabFab HC
C: NabFab LC
D: Divalent metal cation transporter MntH
E: DMT-Nb16_4
F: Anti-Fab nanobody


Theoretical massNumber of molelcules
Total (without water)247,48210
Polymers247,48210
Non-polymers00
Water57632
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16520 Å2
ΔGint-77 kcal/mol
Surface area96270 Å2

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Components

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Antibody , 4 types, 8 molecules HALCNEKF

#1: Antibody NabFab HC


Mass: 25684.463 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Antibody NabFab LC


Mass: 23258.783 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody DMT-Nb16_4


Mass: 14352.809 Da / Num. of mol.: 2
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.: 2
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, 34 molecules BD

#3: Protein Divalent metal cation transporter MntH


Mass: 47054.531 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus capitis (bacteria) / Gene: mntH, NCTC11045_01736 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A4U9TNH6
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 32 / 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 Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody
Type: COMPLEX / Details: Rigid body docked structure / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 8.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34100 / Symmetry type: POINT

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