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Yorodumi- EMDB-13424: Structure of Multidrug and Toxin Compound Extrusion (MATE) transp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13424 | |||||||||
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Title | Structure of Multidrug and Toxin Compound Extrusion (MATE) transporter NorM by NabFab-fiducial assisted cryo-EM | |||||||||
Map data | Map with fulcrum ion center of chimeric nanobody | |||||||||
Sample |
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Function / homology | Multi antimicrobial extrusion protein / MatE / antiporter activity / xenobiotic transmembrane transporter activity / membrane => GO:0016020 / Multidrug resistance protein NorM Function and homology information | |||||||||
Biological species | Vibrio cholerae RC385 (bacteria) / synthetic construct (others) / Lama glama (llama) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.47 Å | |||||||||
Authors | Bloch JS / Mukherjee S / Kowal J / Niederer M / Pardon E / Steyaert J / Kossiakoff AA / Locher KP | |||||||||
Funding support | Switzerland, 1 items
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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 JS / Mukherjee S / Kowal J / Filippova EV / Niederer M / Pardon E / Steyaert J / Kossiakoff AA / Locher KP | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13424.map.gz | 320.6 MB | EMDB map data format | |
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Header (meta data) | emd-13424-v30.xml emd-13424.xml | 15.7 KB 15.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13424_fsc.xml | 16.9 KB | Display | FSC data file |
Images | emd_13424.png | 67.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13424 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13424 | HTTPS FTP |
-Validation report
Summary document | emd_13424_validation.pdf.gz | 362.5 KB | Display | EMDB validaton report |
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Full document | emd_13424_full_validation.pdf.gz | 362.1 KB | Display | |
Data in XML | emd_13424_validation.xml.gz | 14.2 KB | Display | |
Data in CIF | emd_13424_validation.cif.gz | 19.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13424 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13424 | HTTPS FTP |
-Related structure data
Related structure data | 7phpMC 7phqC 7pijC 7rthC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13424.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map with fulcrum ion center of chimeric nanobody | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.67 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of vibrio cholerae MATE transporter NorM, chimeric nanobo...
Entire | Name: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody |
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Components |
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-Supramolecule #1: Complex of vibrio cholerae MATE transporter NorM, chimeric nanobo...
Supramolecule | Name: 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 |
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Source (natural) | Organism: Vibrio cholerae RC385 (bacteria) |
-Macromolecule #1: Multidrug resistance protein NorM
Macromolecule | Name: Multidrug resistance protein NorM / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Vibrio cholerae RC385 (bacteria) |
Molecular weight | Theoretical: 52.462109 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGHHHHHHHH HHSSGLEVLF QGPSGTSVHR YKKEASNLIK LATPVLIASV AQTGMGFVDT IMAGGVSAID MAAVSIAASI WLPSILFGV GLLMALVPVV AQLNGAGRQH KIPFEVHQGL ILALLVSIPI IAVLFQTQFI IRFMDVEEAM ATKTVGYMHA V IFAVPAYL ...String: MGHHHHHHHH HHSSGLEVLF QGPSGTSVHR YKKEASNLIK LATPVLIASV AQTGMGFVDT IMAGGVSAID MAAVSIAASI WLPSILFGV GLLMALVPVV AQLNGAGRQH KIPFEVHQGL ILALLVSIPI IAVLFQTQFI IRFMDVEEAM ATKTVGYMHA V IFAVPAYL LFQALRSFTD GMSLTKPAMV IGFIGLLLNI PLNWIFVYGK FGAPELGGVG CGVATAIVYW IMLLLLLFYI VT SKRLAHV KVFETFHKPQ PKELIRLFRL GFPVAAALFF EVTLFAVVAL LVAPLGSTVV AAHQVALNFS SLVFMFPMSI GAA VSIRVG HKLGEQDTKG AAIAANVGLM TGLATACITA LLTVLFREQI ALLYTENQVV VALAMQLLLF AAIYQCMDAV QVVA AGSLR GYKDMTAIFH RTFISYWVLG LPTGYILGMT NWLTEQPLGA KGFWLGFIIG LSAAALMLGQ RLYWLQKQSD DVQLH LAAK |
-Macromolecule #2: NabFab HC
Macromolecule | Name: NabFab HC / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 25.684463 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV ...String: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH T |
-Macromolecule #3: NabFab LC
Macromolecule | Name: NabFab LC / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 23.258783 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC |
-Macromolecule #4: NorM-Nb17_4
Macromolecule | Name: NorM-Nb17_4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 14.356979 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QRQLVESGGG LVQPGGSLRL SCAASGIIFK INDMGWFRQA PGKEREGVAG ITSGGRTNYA DSVKGRFIIS RDNVKNTVYL QMNSLEPED TAVYYCKSDG LISYAASQLS TYWGKGTPVT VSSHHHHHHE PEA |
-Macromolecule #5: Anti-Fab nanobody
Macromolecule | Name: Anti-Fab nanobody / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 13.390644 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKNT VYLQMNSLK PEDTAVYYCA IDSDTFYSGS YDYWGQGTQV TVSS |
-Macromolecule #6: water
Macromolecule | Name: water / type: ligand / ID: 6 / Number of copies: 21 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 26.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |