+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-17547 | |||||||||
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Title | Structure of 5D3-Fab and nanobody(Nb96)-bound ABCG2 | |||||||||
Map data | ||||||||||
Sample |
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Keywords | MEMBRANE PROTEIN | |||||||||
Function / homology | Function and homology information biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / transepithelial transport / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / NFE2L2 regulating MDR associated enzymes / ABC-type xenobiotic transporter activity / cellular detoxification / Heme biosynthesis / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / protein homodimerization activity / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Mus musculus (house mouse) / Lama glama (llama) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.49 Å | |||||||||
Authors | Irobalieva RN / Manolaridis I / Jackson SM / Ni D / Pardon E / Stahlberg H / Steyaert J / Locher KP | |||||||||
Funding support | Switzerland, 1 items
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Citation | Journal: J Mol Biol / Year: 2023 Title: Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies. Authors: Rossitza N Irobalieva / Ioannis Manolaridis / Scott M Jackson / Dongchun Ni / Els Pardon / Henning Stahlberg / Jan Steyaert / Kaspar P Locher / Abstract: ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and ...ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_17547.map.gz | 9.8 MB | EMDB map data format | |
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Header (meta data) | emd-17547-v30.xml emd-17547.xml | 19.6 KB 19.6 KB | Display Display | EMDB header |
Images | emd_17547.png | 67.5 KB | ||
Others | emd_17547_half_map_1.map.gz emd_17547_half_map_2.map.gz | 152 MB 152 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-17547 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-17547 | HTTPS FTP |
-Validation report
Summary document | emd_17547_validation.pdf.gz | 935.2 KB | Display | EMDB validaton report |
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Full document | emd_17547_full_validation.pdf.gz | 934.8 KB | Display | |
Data in XML | emd_17547_validation.xml.gz | 15.3 KB | Display | |
Data in CIF | emd_17547_validation.cif.gz | 17.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17547 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17547 | HTTPS FTP |
-Related structure data
Related structure data | 8p8jMC 8p7wC 8p8aC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_17547.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.82 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_17547_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_17547_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Structure of nanobody (Nb96)-bound ABCG2
Entire | Name: Structure of nanobody (Nb96)-bound ABCG2 |
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Components |
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-Supramolecule #1: Structure of nanobody (Nb96)-bound ABCG2
Supramolecule | Name: Structure of nanobody (Nb96)-bound ABCG2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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-Supramolecule #2: ATP-binding cassette sub-family G member 2
Supramolecule | Name: ATP-binding cassette sub-family G member 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
-Supramolecule #3: 5D3(Fab) heavy and light chain variable domain
Supramolecule | Name: 5D3(Fab) heavy and light chain variable domain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Mus musculus (house mouse) |
-Supramolecule #4: Nanobody
Supramolecule | Name: Nanobody / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4 |
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Source (natural) | Organism: Lama glama (llama) |
-Macromolecule #1: ATP-binding cassette sub-family G member 2
Macromolecule | Name: ATP-binding cassette sub-family G member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 72.385852 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL ...String: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL GLDKVADSKV GTQFIRGVSG GERKRTSIGM ELITDPSILF LDEPTTGLDS STANAVLLLL KRMSKQGRTI IF SIHQPRY SIFKLFDSLT LLASGRLMFH GPAQEALGYF ESAGYHCEAY NNPADFFLDI INGDSTAVAL NREEDFKATE IIE PSKQDK PLIEKLAEIY VNSSFYKETK AELHQLSGGE KKKKITVFKE ISYTTSFCHQ LRWVSKRSFK NLLGNPQASI AQII VTVVL GLVIGAIYFG LKNDSTGIQN RAGVLFFLTT NQCFSSVSAV ELFVVEKKLF IHEYISGYYR VSSYFLGKLL SDLLP MRML PSIIFTCIVY FMLGLKPKAD AFFVMMFTLM MVAYSASSMA LAIAAGQSVV SVATLLMTIC FVFMMIFSGL LVNLTT IAS WLSWLQYFSI PRYGFTALQH NEFLGQNFCP GLNATGNNPC NYATCTGEEY LVKQGIDLSP WGLWKNHVAL ACMIVIF LT IAYLKLLFLK KYS UniProtKB: Broad substrate specificity ATP-binding cassette transporter ABCG2 |
-Macromolecule #2: 5D3(Fab) light chain variable domain
Macromolecule | Name: 5D3(Fab) light chain variable domain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.594016 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC |
-Macromolecule #3: 5D3(Fab) heavy chain variable domain
Macromolecule | Name: 5D3(Fab) heavy chain variable domain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.843633 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP RGP |
-Macromolecule #4: Nanobody
Macromolecule | Name: Nanobody / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 13.703134 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLQESGGG LVQAGGSLRL SCAASGRSFS SYTMGWFRQA PGKEREFVAA IKWSGDITKY VDSVKGRFTI SRDNSKNTVY LEMNSLKPE DTAVYYCGAD NTWVGYPSSR SSMDYWGQGT QVTVSS |
-Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 72.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V2) / Number images used: 98009 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
-Atomic model buiding 1
Refinement | Space: REAL |
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Output model | PDB-8p8j: |