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- EMDB-13426: Structure of homo-dimeric Staphylococcus capitis divalent metal i... -

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Basic information

Entry
Database: EMDB / ID: EMD-13426
TitleStructure of homo-dimeric Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM
Map data
Sample
  • Complex: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody
    • Protein or peptide: NabFab HC
    • Protein or peptide: NabFab LC
    • Protein or peptide: Divalent metal cation transporter MntH
    • Protein or peptide: DMT-Nb16_4
    • Protein or peptide: Anti-Fab nanobody
  • Ligand: water
KeywordsNabFab / Anti-nanobody Fab / Fiducial / DMT / MEMBRANE PROTEIN
Function / homologyNRAMP family / Natural resistance-associated macrophage protein-like / cadmium ion transmembrane transporter activity / manganese ion transmembrane transporter activity / iron ion transmembrane transport / symporter activity / metal ion binding / plasma membrane / Divalent metal cation transporter MntH
Function and homology information
Biological speciessynthetic construct (others) / Staphylococcus capitis (bacteria) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.45 Å
AuthorsBloch JS / Mukherjee S
Funding support Switzerland, 1 items
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 JS / Mukherjee S / Kowal J / Filippova EV / Niederer M / Pardon E / Steyaert J / Kossiakoff AA / Locher KP
History
DepositionAug 18, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.048
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.048
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7phq
  • Surface level: 0.048
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7phq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13426.png

Downloads & links

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Map

FileDownload / File: emd_13426.map.gz / Format: CCP4 / Size: 5.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.64 Å/pix.
x 112 pix.
= 295.68 Å		2.64 Å/pix.
x 112 pix.
= 295.68 Å		2.64 Å/pix.
x 112 pix.
= 295.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.048 / Movie #1: 0.048
Minimum - Maximum-0.019288054 - 0.100871466
Average (Standard dev.)0.001876177 (±0.009036961)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions112112112
Spacing112112112
CellA=B=C: 295.68002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.642.642.64
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z295.680295.680295.680
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ640640640
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-0.0190.1010.002

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Supplemental data

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Sample components

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Entire : Complex of Staphylococcus capitis divalent metal ion transporter ...

EntireName: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody
Components
  • Complex: Complex of Staphylococcus capitis divalent metal ion transporter (DMT), chimeric nanobody DMTNb16_4, NabFab, and anti-Fab nanobody
    • Protein or peptide: NabFab HC
    • Protein or peptide: NabFab LC
    • Protein or peptide: Divalent metal cation transporter MntH
    • Protein or peptide: DMT-Nb16_4
    • Protein or peptide: Anti-Fab nanobody
  • Ligand: water

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Supramolecule #1: Complex of Staphylococcus capitis divalent metal ion transporter ...

SupramoleculeName: 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
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: NabFab HC

MacromoleculeName: NabFab HC / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.684463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: NabFab LC

MacromoleculeName: NabFab LC / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: Divalent metal cation transporter MntH

MacromoleculeName: Divalent metal cation transporter MntH / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus capitis (bacteria)
Molecular weightTheoretical: 47.054531 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

UniProtKB: Divalent metal cation transporter MntH

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Macromolecule #4: DMT-Nb16_4

MacromoleculeName: DMT-Nb16_4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.352809 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QRQLVESGGG LVQPGGSLRL SCAASRSIFS IDTAGWFRQA PGKEREGVAT ITRDGNANYA DSVKGRFTIS RDRARNTVYL QMNSLEPED TAVYYCNAAI RTTVRTSAQE YWGKGTPVTV SSHHHHHHEP E

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Macromolecule #5: Anti-Fab nanobody

MacromoleculeName: Anti-Fab nanobody / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.390644 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKNT VYLQMNSLK PEDTAVYYCA IDSDTFYSGS YDYWGQGTQV TVSS

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 32 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 26.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34100
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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