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Growth conditions Synechocystis sp. PCC 6803 (ref.
7) was grown photoheterotrophically in mineral medium BG-11 (ref.
16), containing kanamycin and glucose at 30C, and with illumination
of 70 mmol photonsm-2 s-1. Iron-stressed cultures were obtained
by inoculating the same BG-11 medium lacking iron-containing compounds.
Cells were harvested after 3 d, and thylakoid membranes isolated
according to a method similar to that described in ref. 17. The
thylakoid membranes (1 mg chl/ml) were then solubilized with 1%
b-D-dodecyl maltoside at 4 8C for 10 min. The solubilized membranes
were centrifuged at 150,000g using a Beckman Ti70 rotor. The supernatant
was then passed through a Ni2+-affinity column.-PSII was selectively
bound to the column via the His-tag while the non-bound fraction
containing-PSI was collected and subjected to sucrose density centrifugation.
Biochemical characterization SDS-PAGE and western blotting were
performed as described in ref. 18. Optical absorption spectra were
measured at room temperature using a Shimadzu MPS 2000 spectrometer.
Steady-state fluorescence spectra were obtained using a Perkin Elmer
LS50 at 77K and measured with an excitation wavelength of 440 nm.
To record excitation spectra, the sample was excited between 650
and 700nm and the fluorescence detected at 720 nm. HPLC size exclusion
analyses were performed using a Phenomenex BioSep SEC S3000 column.
Electron microscopy Preparations were negatively stained
with 2% uranyl acetate on glow discharged carbon evaporated grids,
and imaged using a Philips CM 100 electron microscope at 80 kV.
The magnification was calibrated to be ´51,500. Twenty electron
micrographs were taken for each preparation, and subsequently calculated
to have the first minima of contrast transfer function in the range
of 17-23 Angstrom .
Image processing and modelling Electron micrographs were
digitized using a Leafscan 45 densitometer set at a step size of
10 um. Single-particle data sets of approximately 3,000 (CP43'-PSI
supercomplex) and 4,000 (PSI trimer) were obtained by interactively
selecting all possible particles from the micrographs. All subsequent
processing was performed within the IMAGIC-5 software environment19,20.
The single-particle images were coarsened by a factor of 2 resulting
in 3.88 Angstrom per pixel on the specimen scale. Reference free
alignment coupled with multivariate statistical classification21
was used to identify initial class averages, which were then used
for iterative refinement until the data merged, resulting in the
improved class averages shown. Co-ordinate data sets were obtained
from the RCSB data bank (http://www.rcsb.org) under the entry codes
1C51 (PSI 4 Angstrom structure10) and 1FE1 (PSII 3.8 Angstrom structure11).
These structural models were visualized using the program Swiss-PDB
viewer22 (Glaxo-Wellcome Experimental Research) and overlaid at
the same scale onto the calculated singleparticle projection maps.
The carbon-a backbone for the transmembrane helices of the CP43
subunit was extracted from the 1FE1 co-ordinates and modelled into
each subunit of the ring surrounding the-PSI trimer, according to
the centre of mass observed for each of the 18 subunits within the
ring.
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