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- EMDB-42793: Aquaporin Z with ALFA tag and bound to nanobody -

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Basic information

Entry
Database: EMDB / ID: EMD-42793
TitleAquaporin Z with ALFA tag and bound to nanobody
Map dataEM map
Sample
  • Complex: Aquaporin Z with ALFA tag bound to nanobody
    • Complex: Aquaporin Z with ALFA tag
      • Protein or peptide: Aquaporin Z
    • Complex: nanobody
      • Protein or peptide: anti-ALFA nanobody
  • Ligand: CARDIOLIPIN
KeywordsAqpZ / water channel / ALFA tag / cardiolipin / MEMBRANE PROTEIN
Function / homology
Function and homology information


intracellular water homeostasis / water transport / water channel activity / response to osmotic stress / identical protein binding / plasma membrane
Similarity search - Function
Aquaporin Z / Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsStover L / Bahramimoghaddam H / Wang L / Zhou M / Laganowsky A
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM138863 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM139876 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RM1GM145416 United States
CitationJournal: J Struct Biol X / Year: 2024
Title: Grafting the ALFA tag for structural studies of aquaporin Z.
Authors: Lauren Stover / Hanieh Bahramimoghaddam / Lie Wang / Samantha Schrecke / Gaya P Yadav / Ming Zhou / Arthur Laganowsky /
Abstract: Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate ...Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate structure determination of membrane proteins, such as the use of antibodies. Here, we graft onto AqpZ the ALFA tag (AqpZ-ALFA), an alpha helical epitope, to make use of the high-affinity anti-ALFA nanobody (nB). Native mass spectrometry reveals the AqpZ-ALFA fusion forms a stable, 1:1 complex with nB. Single-particle cryogenic electron microscopy studies reveal the octameric (AqpZ-ALFA)(nB) complex forms a dimeric assembly and the structure was determined to 1.9 Å resolution. Dimerization of the octamer is mediated through stacking of the symmetrically bound nBs. Tube-like density is also observed, revealing a potential cardiolipin binding site. Grafting of the ALFA tag, or other epitope, along with binding and association of nBs to promote larger complexes will have applications in structural studies and protein engineering.
History
DepositionNov 13, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42793.png

Downloads & links

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Map

FileDownload / File: emd_42793.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 350 pix.
= 291.2 Å		0.83 Å/pix.
x 350 pix.
= 291.2 Å		0.83 Å/pix.
x 350 pix.
= 291.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.145
Minimum - Maximum-0.27925134 - 0.87088656
Average (Standard dev.)-0.0011669776 (±0.028166246)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 291.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Aquaporin Z with ALFA tag bound to nanobody

EntireName: Aquaporin Z with ALFA tag bound to nanobody
Components
  • Complex: Aquaporin Z with ALFA tag bound to nanobody
    • Complex: Aquaporin Z with ALFA tag
      • Protein or peptide: Aquaporin Z
    • Complex: nanobody
      • Protein or peptide: anti-ALFA nanobody
  • Ligand: CARDIOLIPIN

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Supramolecule #1: Aquaporin Z with ALFA tag bound to nanobody

SupramoleculeName: Aquaporin Z with ALFA tag bound to nanobody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 161 KDa

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Supramolecule #2: Aquaporin Z with ALFA tag

SupramoleculeName: Aquaporin Z with ALFA tag / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: Aquaporin Z

MacromoleculeName: Aquaporin Z / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 26.710988 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFRKLAAECF GTFWLVFGGC GSAVLAAGFP ELGIGFAGVA LAFGLTVLTM AFAVGHISGG HFNPAVTIGL WAGGRFPAKE VVGYVIAQV VGGIVAAALL YLIASGKTGF DAAASGFASN GYGEHSPGGY SMLSALVVEL VLSAGFLLVI HGATDKFAPA G FAPIAIGL ...String:
MFRKLAAECF GTFWLVFGGC GSAVLAAGFP ELGIGFAGVA LAFGLTVLTM AFAVGHISGG HFNPAVTIGL WAGGRFPAKE VVGYVIAQV VGGIVAAALL YLIASGKTGF DAAASGFASN GYGEHSPGGY SMLSALVVEL VLSAGFLLVI HGATDKFAPA G FAPIAIGL ALTLIHLISI PVTNTSVNPA RSTAVAIFQG GWALEQLWFF WVVPIVGGII GGLIYRTLLR ASRLEEELRR RL TEPGGGP GASWSHPQFE K

UniProtKB: Aquaporin Z

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Macromolecule #2: anti-ALFA nanobody

MacromoleculeName: anti-ALFA nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.599183 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGEVQLQESG GGLVQPGGSL RLSCTASGVT ISALNAMAMG WYRQAPGERR VMVAAVSERG NAMYRESVQG RFTVTRDFTN KMVSLQMDN LKPEDTAVYY CHVLEDRVDS FHDYWGQGTQ VTVSS

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Macromolecule #3: CARDIOLIPIN

MacromoleculeName: CARDIOLIPIN / type: ligand / ID: 3 / Number of copies: 8 / Formula: CDL
Molecular weightTheoretical: 1.464043 KDa
Chemical component information

ChemComp-CDL:
CARDIOLIPIN / phospholipid*YM

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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

Concentration10 mg/mL
BufferpH: 7.4 / Details: 20mM TRIS pH 7.4, 100mM NaCl, and 0.5% C8E4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1785869
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1rc2

Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.0) / Number images used: 918328
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.3.0)

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Atomic model buiding 1

Initial modelPDB ID:

1rc2


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8uy6:
Aquaporin Z with ALFA tag and bound to nanobody

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