Supplementary Information and Figure Legends
(Nakagawa et al “Structure and different conformational states of native AMPA
receptor complexes”)
Purification of AMPA-R and Fab labeling
150 - 300 adult rat brains were homogenized in 20 mM HEPES, pH 7.4, 320 mM
sucrose, 5 mM EDTA, 5 mM EGTA, 30 M NBQX supplemented with protease
inhibitors (1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupetin, 1 g/ml pepstatin, and
500 M benzamidine). Supernatant obtained by centrifuging the homogenate at 3,000 g
for 15 min was further spun at 38,400 g for 15 min to obtain a membrane pellet (P2
fraction). P2 was resuspended in SB1 (20 mM HEPES, pH 7.4, 1 M KI, 5 mM EDTA, 5
mM EGTA, and 30 M NBQX) and membranes were collected by centrifugation.
Membranes were further washed with SB2 (20 mM HEPES, pH 7.4, 4 M urea, 5 mM
EDTA, 5 mM EGTA, 30 M NBQX) to remove associated proteins and collected again
by centrifugation. Finally, membranes were solublized in RB (20 mM HEPES, pH 7.4,
100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 % CHAPS, 30 M NBQX, with protease
inhibitors) for 30 min with gentle stirring and ultracentrifuged at 100,000 g to remove
insoluble material. The final supernatant was applied to an anti-GluR2 C-terminus
antibody affinity column (0.5 ml bed volume, antibody concentration 2 mg/ml). After
washing the column with 3 ml of sample buffer, GluR2 containing AMPA-Rs were
eluted with 20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1%
CHAPS, 30 M NBQX, 0.5 mg/ml GluR2 C-terminal epitope peptide
(GYNVYGIESVKI). The proteins were further purified on a Superdex 200 gel filtration
column (Pharmacia) in 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM
EDTA, 0.3 mM ZnSO4, 1% CHAPS. Zinc was added in the final buffer because it is
found in the dimer interface of the ligand binding domain crystals1. When indicated 1%
CHAPS was substituted by 0.1% dodecyl maltoside or 0.6% decyl maltoside. Western
blotting (Supplementary Fig. 1b) and mass spectrometry analysis (not shown) confirmed
the presence of AMPA-R subunits in the ~100 kDa band. The proteins GRIP 2,3 and
SAP97 4, which bind to the cytoplasmic tails of AMPA-Rs, were absent in the final
preparation (Supplementary Fig. 1b).
Polyclonal antibodies against the following peptides were raised in rabbits and
affinity-purified with the antigen peptides. C-terminus of GluR1 (SHSSGMPLGATGL),
C-terminus of GluR2 (EGYNVYGIESVKI), NTD of GluR1 (RTSDSRDHTRVDWKR),
pan-TARP#1 (CIQKDSKDSLHANTANRRTTPV), and pan-TARP#2
(CPEDADYEADTAEYFLR). Fab fragments were purified using the Immunopure IgG1
F(ab’) and the F(ab’)2 Fab purification kit (Pierce) followed by gel filtration on a
Superdex 200 column (Pharmacia). Labelling was performed by incubating AMPA-Rs
with Fab fragments at a molar ratio of 1:4 to 1:8 overnight at 4oC in 20 mM HEPES, pH
7.4, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% CHAPS.
Image processing
Electron micrographs were digitized with a SCAI scanner (Zeiss) using a step size of 7
m. 3 x 3 pixels were averaged yielding a pixel size on the specimen level of 4.04 Å for
the negatively stained and 5 Å for the cryo-negatively stained specimens. Image
processing was performed with the SPIDER software package 5. For conventional
negatively stained specimens, particles were selected interactively using WEB, the
display program associated with SPIDER, and windowed into small images of 100 x 100
pixels. Projection averages were calculated over 10 cycles of K-means classification and
multireference alignment specifying 100 output classes. For 3D reconstructions of the
cryo-negatively stained preparations, particle pairs were interactively selected from a
total of 373 image pairs of 50o/0o tilted specimens and windowed into small images of
100 x 100 pixels. Table 1 summarizes the number of particles used in this study. The
particles from the images of the untilted specimens were used for classification into 100
classes as before. The images of the tilted specimens in each class were used to calculate
initial 3D reconstructions of individual classes by backprojection, backprojection
refinement, and angular refinement. The final volume obtained by angular refinement
with SPIDER was used as input model for FREALIGN, which was used for further
refinement of the orientational parameters of the individual particles and to correct for the
contrast transfer function of each particle image according to its defocus value 6. The
correct defocus value for each particle image was deduced from the position of each
particle in the image and the tilt angles and defocus values of the images, which were
determined with CTFTILT 7. Electron micrographs showing a high degree of
astigmatism were rejected. Particles selected from both the images of the tilted and the
untilted specimens were used for the refinement with FREALIGN. The resolution of the
final 3D reconstructions were estimated using Fourier shell correlation (FSC) and ranged
from 42 to 46 Å using the FSC = 0.5 criterion or from 31 to 33 Å using the FSC = 0.142
criterion proposed by Rosenthal and Henderson 8.
Supplementary Figure 1. Purification of AMPA-Rs.
a. Fractions eluting from the anti-GluR2 immuno-affinity column (“AMPA crude”) and
the subsequent gel filtration (Superdex 200) column (fractions 13 – 21) were resolved by
SDS-PAGE (4 – 15 % gradient gel) and visualized by silver staining. The flow rate was
0.5 ml/min and 0.5 ml fractions were collected. Fraction 17 was used for EM.
b. Fractions shown in (a) were immunoblotted for the proteins indicated on the left.
Brain extract (3,000 g centrifugation supernatant of brain homogenate) was used as
positive controls for immunoblots against GRIP and SAP97.
Supplementary Figure 2. Plot of the angle distributions and comparison of
reprojections from 3D models with the corresponding raw particle images.
a. Distribution of the angles (phi and theta) assigned by FREALIGN to the particle
images used for the 3D reconstruction shown at the left bottom of each plot. Theta is
zero at the center and 90o at the outer edge of the circle. Phi is zero at the position
indicated in the figure and increases radially in counter-clockwise direction. The
distribution is consistent with the random conical tilt reconstruction approach, where the
majority of particles are distributed around phi = 50o and the images from the untilted
specimens (phi = 0o) are concentrated in the center of the plot.
b. Representative side-by-side comparisons of raw particle images (left panels) with the
corresponding reprojections from the final 3D reconstructions (right panels).
Supplementary Figure 3. 3D density map of AMPA-R in the type I conformation
filtered to 42 Å (FSC = 0.5 criterion) or 31 Å (FSC = 0.142 criterion).
The difference between density maps filtered to a resolution of 31 Å or 42 Å is virtually
undetectable for a particle the size of AMPA-R and makes no differencce for the
placement of crystal structures into the density map. We therefore filtered the density
maps shown in Fig. 2 to the more conservative resolution value of 42 Å.
Supplementary Figure 4. ClustalW alignment of mGluR1 (extracellular domain),
LIVBP, and GluR2-NTD.
The amino acid residues present in the extracellular domain of mGluR1 but not in GluR2
are highlighted in yellow. These residues were removed from the crystal structure that
was placed into the EM density map in Fig. 2.
Supplementaty Figure 5. Class averages of particles obtained under different drug
treatments.
Negative stain EM images of particles from the same preparation, left untreated (a),
treated with 1 mM glutamate (b), or with 1 mM glutamate plus 330 M CTZ (c).
Particles were classified into 100 classes by multivariate classification (K-means
classification) and multi-reference alignment. The orientations of each class average
image are not the same. Note the lack of clear Type I particles in the glutamate treated
class averages (b). To quantify the change of the distribution of the conformation we
assigned each class average as “unclassified” (O), “Type I” (I), or “Type II” (II). The
number of particles contained in each class average is indicated in the bottom right of
each box.
Supplementary Table 1. Peptides and corresponding proteins identified by
LC/MS/MS tandem mass spectrometry analysis of the bands indicated by asterisks
in Fig. 3a.
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