Study
Membrane protein
(MW)
Amphipol
EM
methods
Resolution
(Å)
Main observations relevant to the use of amphipols for EM
References
12.27
Mitochondrial
respirasome from
bovine mitochondrion
(~1.7 MDa)
A8-35
Cryo-EM
9.1–10.4
A sequel to Study 12.5, where the resolution was pushed to ~9-Å resolution. Sample preparation was improved by performing the initial purification in the detergent PCC-a-M, rather than
digitonin, before transfer to A8-35, yielding biochemically more homogeneous samples, the
images of which could be distributed into three classes, whose structures were solved
respectively to 9.1, 10.4, and 9.9-Å resolution. The improved structure shows that most
protein-protein contacts between complexes I, III, and IV in the membrane are mediated by
supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III
dimer, one is resolved, indicating that this domain is immobile and therefore unable to transfer
electrons, The functional asymmetry of complex III provides strong evidence for directed
electron flow from complex I to complex IV through the active complex III monomer.
Sousa et al.
(2016)
12.28
HasR heme transporter
and HasR/HasA complex from Serratia
marcescens.
(~95 and ~115 kDa)
A8-35
Negative
staining
20
HasR, a β-barrel MP from the outer membrane of Serratia marcescens, imports heme delivered by
HasA, an extracellular heme carrier protein. Following trapping with A8-35 and negative staining,
the structures of HasR and the HasR/HasA complex were solved to 20-Å resolution, revealing
that the HasR periplasmic domain responsible for signal transfer i) is highly flexible in two stages
of signalling; ii) extends into the periplasm approximately 70–90 Å away from the TM β-barrel;
and iii) exhibits local conformational changes in response to the binding of heme-loaded HasA.
Wojtowicz
et al. (2016)
12.29
Rabbit transient
receptor potential
vanilloid cation
channel 2 (TRPV2)
(~280 kDa)
A8-35
Cryo-EM
~4
TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. It has been implicated in many biological functions,
including somatosensation, osmosensation, and innate immunity. TRPV2 was transferred from
a DDM/lipid mixture to A8-35 using Bio-Beads. Cryo-EM single-particle images were analyzed
to a resolution of ~4 Å, with regions resolved to 3.3 Å. Comparison with the TRPV1 structures
suggests mechanisms for activation and desensitization. Putative bound lipids are identified.
Zubcevic
et al. (2016)
12.30
Caenorhabditis
elegans cyclicnucleotide-gated
channel (TAX-4)
A8-35
Cryo-EM
3.5
Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the
voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic
nucleotides instead of transmembrane voltage. The structure of a cyclic-nucleotide-gated
channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound
open state trapped in A8-35 has been solved to 3.5-Å-resolution by single-particle cryo-EM.
Li et al. (2017)
12.31
Pneumolysin pore
(Streptococcus
pneumoniae)
(~2.2 MDa)
A8-35
Cryo-EM
4.5
Pneumolysin, the main virulence factor from Streptococcus pneumoniae, forms large pores in
the plasma membrane of target cells, resulting in cell lysis. The toxin was expressed under its
soluble, monomeric form, incubated with liposomes, and the resulting proteoliposomes solubilized in Cymal-6. Following addition of A8-35, the detergent was removed by dialysis.
Cryo-EM images were analyzed to 4.5-Å resolution. The pores comprise 42 monomers
assembled in a ring, each monomer featuring four TM β -strands, resulting in a 168-strand,
260-Å-wide
β-barrel. A model for the step-wise transition from the soluble monomer to the
final pore is proposed.
van Pee et al.
(2017)
12.32
AcrABZ-TolC
multidrug efflux pump
(E. coli)
(~770 kDa)
A8-35
Cryo-EM
3.6–6.5
Bacterial efflux pumps confer multidrug resistance by expelling diverse antibiotics from the
cell. The components of the AcrABZ-TolC pump were co-expressed and the complex solubilized and purified in DDM solution. The purified complex was supplemented with A8-35
and the detergent removed using Bio-Beads. Cryo-EM images of AcrABZ-TolC in the presence or absence of an inhibitor were analyzed respectively to 3.6-Å and 6.5-Å resolution
(FSC = 0.143). Comparison of the two models reveals a quaternary structural switch that
allosterically couples and synchronizes initial ligand binding with channel opening.
Wang et al.
(2017)
12.33
Human transient
receptor potential
channel polycystin-2
(PC2)
(~440 kDa)
A8-35
Cryo-EM
4.2–4.3
Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca 2+
signaling
pathways. PC2 was expressed in HEK cells and solubilized and purified in LMNG/cholesterol
hemisuccinate solutions. Purified PC2 was supplemented with A8-35 and the detergent removed
using Bio-Beads. Analysis of cryo-EM images yielded two conformations, with resolutions of 4.2
and 4.3 Å, corresponding to a more or less open pore binding more or less Ca 2+
ions.
Wilkes et al.
(2017)
548
12 The Use of Amphipols for Electron Microscopy
Membrane protein
(MW)
Amphipol
EM
methods
Resolution
(Å)
Main observations relevant to the use of amphipols for EM
References
12.27
Mitochondrial
respirasome from
bovine mitochondrion
(~1.7 MDa)
A8-35
Cryo-EM
9.1–10.4
A sequel to Study 12.5, where the resolution was pushed to ~9-Å resolution. Sample preparation was improved by performing the initial purification in the detergent PCC-a-M, rather than
digitonin, before transfer to A8-35, yielding biochemically more homogeneous samples, the
images of which could be distributed into three classes, whose structures were solved
respectively to 9.1, 10.4, and 9.9-Å resolution. The improved structure shows that most
protein-protein contacts between complexes I, III, and IV in the membrane are mediated by
supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III
dimer, one is resolved, indicating that this domain is immobile and therefore unable to transfer
electrons, The functional asymmetry of complex III provides strong evidence for directed
electron flow from complex I to complex IV through the active complex III monomer.
Sousa et al.
(2016)
12.28
HasR heme transporter
and HasR/HasA complex from Serratia
marcescens.
(~95 and ~115 kDa)
A8-35
Negative
staining
20
HasR, a β-barrel MP from the outer membrane of Serratia marcescens, imports heme delivered by
HasA, an extracellular heme carrier protein. Following trapping with A8-35 and negative staining,
the structures of HasR and the HasR/HasA complex were solved to 20-Å resolution, revealing
that the HasR periplasmic domain responsible for signal transfer i) is highly flexible in two stages
of signalling; ii) extends into the periplasm approximately 70–90 Å away from the TM β-barrel;
and iii) exhibits local conformational changes in response to the binding of heme-loaded HasA.
Wojtowicz
et al. (2016)
12.29
Rabbit transient
receptor potential
vanilloid cation
channel 2 (TRPV2)
(~280 kDa)
A8-35
Cryo-EM
~4
TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. It has been implicated in many biological functions,
including somatosensation, osmosensation, and innate immunity. TRPV2 was transferred from
a DDM/lipid mixture to A8-35 using Bio-Beads. Cryo-EM single-particle images were analyzed
to a resolution of ~4 Å, with regions resolved to 3.3 Å. Comparison with the TRPV1 structures
suggests mechanisms for activation and desensitization. Putative bound lipids are identified.
Zubcevic
et al. (2016)
12.30
Caenorhabditis
elegans cyclicnucleotide-gated
channel (TAX-4)
A8-35
Cryo-EM
3.5
Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the
voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic
nucleotides instead of transmembrane voltage. The structure of a cyclic-nucleotide-gated
channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound
open state trapped in A8-35 has been solved to 3.5-Å-resolution by single-particle cryo-EM.
Li et al. (2017)
12.31
Pneumolysin pore
(Streptococcus
pneumoniae)
(~2.2 MDa)
A8-35
Cryo-EM
4.5
Pneumolysin, the main virulence factor from Streptococcus pneumoniae, forms large pores in
the plasma membrane of target cells, resulting in cell lysis. The toxin was expressed under its
soluble, monomeric form, incubated with liposomes, and the resulting proteoliposomes solubilized in Cymal-6. Following addition of A8-35, the detergent was removed by dialysis.
Cryo-EM images were analyzed to 4.5-Å resolution. The pores comprise 42 monomers
assembled in a ring, each monomer featuring four TM β -strands, resulting in a 168-strand,
260-Å-wide
β-barrel. A model for the step-wise transition from the soluble monomer to the
final pore is proposed.
van Pee et al.
(2017)
12.32
AcrABZ-TolC
multidrug efflux pump
(E. coli)
(~770 kDa)
A8-35
Cryo-EM
3.6–6.5
Bacterial efflux pumps confer multidrug resistance by expelling diverse antibiotics from the
cell. The components of the AcrABZ-TolC pump were co-expressed and the complex solubilized and purified in DDM solution. The purified complex was supplemented with A8-35
and the detergent removed using Bio-Beads. Cryo-EM images of AcrABZ-TolC in the presence or absence of an inhibitor were analyzed respectively to 3.6-Å and 6.5-Å resolution
(FSC = 0.143). Comparison of the two models reveals a quaternary structural switch that
allosterically couples and synchronizes initial ligand binding with channel opening.
Wang et al.
(2017)
12.33
Human transient
receptor potential
channel polycystin-2
(PC2)
(~440 kDa)
A8-35
Cryo-EM
4.2–4.3
Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca 2+
signaling
pathways. PC2 was expressed in HEK cells and solubilized and purified in LMNG/cholesterol
hemisuccinate solutions. Purified PC2 was supplemented with A8-35 and the detergent removed
using Bio-Beads. Analysis of cryo-EM images yielded two conformations, with resolutions of 4.2
and 4.3 Å, corresponding to a more or less open pore binding more or less Ca 2+
ions.
Wilkes et al.
(2017)
548
12 The Use of Amphipols for Electron Microscopy