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Yorodumi- PDB-7pij: Structure of Staphylococcus capitis divalent metal ion transporte... -
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-Basic information
Entry | Database: PDB / ID: 7pij | ||||||
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Title | Structure of Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / NabFab / Anti-nanobody Fab / Fiducial / DMT | ||||||
Function / homology | metal ion transmembrane transporter activity / NRAMP family / Natural resistance-associated macrophage protein-like / symporter activity / membrane => GO:0016020 / metal ion binding / plasma membrane / Divalent metal cation transporter MntH Function and homology information | ||||||
Biological species | synthetic construct (others) Staphylococcus capitis (bacteria) Lama glama (llama) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å | ||||||
Authors | Bloch, J.S. / Mukherjee, S. / Kowal, J. / Kossiakoff, A.A. / Locher, K.P. | ||||||
Funding support | Switzerland, 1items
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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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7pij.cif.gz | 197.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7pij.ent.gz | 159.8 KB | Display | PDB format |
PDBx/mmJSON format | 7pij.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7pij_validation.pdf.gz | 619.3 KB | Display | wwPDB validaton report |
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Full document | 7pij_full_validation.pdf.gz | 636 KB | Display | |
Data in XML | 7pij_validation.xml.gz | 41.6 KB | Display | |
Data in CIF | 7pij_validation.cif.gz | 60.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pi/7pij ftp://data.pdbj.org/pub/pdb/validation_reports/pi/7pij | HTTPS FTP |
-Related structure data
Related structure data | 13438MC 7phpC 7phqC 7rthC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Antibody , 4 types, 4 molecules HLNK
#1: Antibody | 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) |
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#2: Antibody | 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 | Mass: 14423.888 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 | 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) |
-Protein / Non-polymers , 2 types, 17 molecules B
#3: Protein | Mass: 47054.531 Da / Num. of mol.: 1 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 |
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#6: Water | ChemComp-HOH / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 26 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 433559 / Symmetry type: POINT |