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- EMDB-13438: Structure of Staphylococcus capitis divalent metal ion transporte... -

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Basic information

Entry
Database: EMDB / ID: EMD-13438
TitleStructure of 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: DMTNb16_4
    • Protein or peptide: Anti-Fab nanobody
  • Ligand: water
Function / homologyNRAMP family / Natural resistance-associated macrophage protein-like / metal ion transmembrane transporter activity / 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: 3.78 Å
AuthorsBloch JS / Mukherjee S / Kowal J / Kossiakoff AA / Locher KP
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 20, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.365
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.365
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7pij
  • Surface level: 0.365
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13438.png

Downloads & links

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Map

FileDownload / File: emd_13438.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 448 pix.
= 295.68 Å		0.66 Å/pix.
x 448 pix.
= 295.68 Å		0.66 Å/pix.
x 448 pix.
= 295.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.365 / Movie #1: 0.365
Minimum - Maximum-1.9206635 - 2.8468273
Average (Standard dev.)0.001910869 (±0.03609872)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 295.68002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.660.660.66
M x/y/z448448448
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/NS448448448
D min/max/mean-1.9212.8470.002

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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: DMTNb16_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
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: 1 / 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: 1 / 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: 1 / 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

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Macromolecule #4: DMTNb16_4

MacromoleculeName: DMTNb16_4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.423888 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QRQLVESGGG LVQPGGSLRL SCAASRSIFS IDTAGWFRQA PGKEREGVAT ITRDGNANYA DSVKGRFTIS RDRARNTVYL QMNSLEPED TAVYYCNAAI RTTVRTSAQE YWGKGTPVTV SSHHHHHHEP EA

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Macromolecule #5: Anti-Fab nanobody

MacromoleculeName: Anti-Fab nanobody / type: protein_or_peptide / ID: 5 / Number of copies: 1 / 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: 16 / 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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 433559
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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