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- EMDB-13426: Structure of homo-dimeric Staphylococcus capitis divalent metal i... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-13426 | |||||||||
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Title | Structure of homo-dimeric Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM | |||||||||
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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![]() | |||||||||
Biological species | synthetic construct (others) / ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.45 Å | |||||||||
![]() | Bloch JS / Mukherjee S / Kowal J / Kossiakoff AA / Locher KP | |||||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.7 KB 15.7 KB | Display Display | ![]() |
Images | ![]() | 32.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7phqMC ![]() 7phpC ![]() 7pijC ![]() 7rthC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Voxel size | X=Y=Z: 2.64 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Complex of Staphylococcus capitis divalent metal ion transporter ...
Entire | Name: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody |
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Components |
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-Supramolecule #1: Complex of Staphylococcus capitis divalent metal ion transporter ...
Supramolecule | Name: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 / Details: Rigid body docked structure |
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Source (natural) | Organism: synthetic construct (others) |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: NabFab HC
Macromolecule | Name: NabFab HC / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 25.684463 KDa |
Recombinant expression | Organism: ![]() ![]() |
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 #2: NabFab LC
Macromolecule | Name: NabFab LC / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 23.258783 KDa |
Recombinant expression | Organism: ![]() ![]() |
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 #3: Divalent metal cation transporter MntH
Macromolecule | Name: Divalent metal cation transporter MntH / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 47.054531 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MSVAVGYMDP GNWITSMQGG AQYGYTLLFV ILISSLAAML LQSMTVRLGI ATGKDLAQMT RHFLSKPVAI IFWIIAELAI IATDIAEVI GSAIALDLIF GIPLIVGALI TVFDVFLLLF IMRFGFRKIE AIVGTLIFTV LAIFVFEVFI SSPQLTDILN G FVPHKEIV ...String: MSVAVGYMDP GNWITSMQGG AQYGYTLLFV ILISSLAAML LQSMTVRLGI ATGKDLAQMT RHFLSKPVAI IFWIIAELAI IATDIAEVI GSAIALDLIF GIPLIVGALI TVFDVFLLLF IMRFGFRKIE AIVGTLIFTV LAIFVFEVFI SSPQLTDILN G FVPHKEIV TNQGILYIAL GIIGATIMPH NLYLHSSIVQ SRKYDRHDNQ EKAQAIKYAT IDSNLQLSIA FVVNCLLLTL GA ALFFGTK TNDLGGFYDL YHALKTEPVL GATLGGIMST LFAVALLASG QNSTITGTLA GQIVMEGFLR LSIPNWLRRL ITR ALAVIP VIICLIIFKG NSEKIEQLLV FSQVFLSIAL PFSLIPLQLA TSNKKLMGPF INKTWVNIIS WTLIVILSGL NVYL IIQTF QELALEVLFQ GPHHHHHHHH HH |
-Macromolecule #4: DMT-Nb16_4
Macromolecule | Name: DMT-Nb16_4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 14.352809 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: QRQLVESGGG LVQPGGSLRL SCAASRSIFS IDTAGWFRQA PGKEREGVAT ITRDGNANYA DSVKGRFTIS RDRARNTVYL QMNSLEPED TAVYYCNAAI RTTVRTSAQE YWGKGTPVTV SSHHHHHHEP E |
-Macromolecule #5: Anti-Fab nanobody
Macromolecule | Name: Anti-Fab nanobody / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 13.390644 KDa |
Recombinant expression | Organism: ![]() ![]() |
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: 32 / 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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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE-PROPANE |
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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: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 8.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34100 |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |