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

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Basic information

Entry
Database: PDB / ID: 8uy6
TitleAquaporin Z with ALFA tag and bound to nanobody
Components
  • Aquaporin Z
  • anti-ALFA nanobody
KeywordsMEMBRANE PROTEIN / AqpZ / water channel / ALFA tag / cardiolipin
Function / homology
Function and homology information


intracellular water homeostasis / water channel activity / water transport / 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
CARDIOLIPIN / Aquaporin Z
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsStover, L. / Bahramimoghaddam, H. / Wang, L. / Zhou, M. / Laganowsky, A.
Funding support United States, 3items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Aquaporin Z
B: anti-ALFA nanobody
C: Aquaporin Z
D: anti-ALFA nanobody
E: Aquaporin Z
F: anti-ALFA nanobody
G: Aquaporin Z
H: anti-ALFA nanobody
I: Aquaporin Z
J: anti-ALFA nanobody
K: Aquaporin Z
L: anti-ALFA nanobody
M: Aquaporin Z
N: anti-ALFA nanobody
O: Aquaporin Z
P: anti-ALFA nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)334,19424
Polymers322,48116
Non-polymers11,7128
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Aquaporin Z / Bacterial nodulin-like intrinsic protein / Water channel AqpZ


Mass: 26710.988 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: aqpZ, bniP, b0875, JW0859 / Plasmid: pET15 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-A1 / References: UniProt: P60844
#2: Antibody
anti-ALFA nanobody


Mass: 13599.183 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pET29 / Production host: Escherichia coli (E. coli) / Strain (production host): Shuffle T7 Express
#3: Chemical
ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C81H156O17P2 / Comment: phospholipid*YM
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Aquaporin Z with ALFA tag bound to nanobodyCOMPLEX#1-#20MULTIPLE SOURCES
2Aquaporin Z with ALFA tagCOMPLEX#11RECOMBINANT
3nanobodyCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.161 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.4 / Details: 20mM TRIS pH 7.4, 100mM NaCl, and 0.5% C8E4
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.0particle selection
4cryoSPARC4.3.0CTF correction
7Coot0.9.8model fitting
9cryoSPARC4.3.0initial Euler assignment
10cryoSPARC4.3.0final Euler assignment
11cryoSPARC4.3.0classification
12cryoSPARC4.3.03D reconstruction
13PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1785869
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 918328 / Symmetry type: POINT
Atomic model buildingPDB-ID: 1RC2

1rc2


Pdb chain-ID: A / Accession code: 1RC2 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323112
ELECTRON MICROSCOPYf_angle_d0.52131216
ELECTRON MICROSCOPYf_dihedral_angle_d10.7183736
ELECTRON MICROSCOPYf_chiral_restr0.0423448
ELECTRON MICROSCOPYf_plane_restr0.0043912

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