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Isolation and biochemical characterization
Prochlorococcus sp. MIT 9313 (ref. 2) was grown in PCR-S11 medium1.
Iron-stressed cultures were obtained by two consecutive transfers
of the cells into fresh medium containing only 1% of the standard
FeCl3 concentration of the PCR-SII medium but having the normal
Na2-EDTA concentration. Cells in the exponential phase of growth
were collected and thylakoid membranes isolated according to ref.
15. The thylakoids (1 mg chlorophyll ml-1) were then solubilized
with 1% b-D-dodecyl maltoside at 4 8C for 10 min. The solubilized
membranes were centrifuged at 100,000g using a Beckman Ti70 rotor
and the solubilized fraction subjected to sucrose density centrifugation6.
The resulting bands were independently removed for biochemical and
structural characterization. SDS–PAGE and western blotting
were performed according to ref. 16. Antibodies were a gift from
P. Nixon (anti-D1) and J. Golbeck (anti-PsaC). N-terminal sequencing
of proteins was performed by J. Keen.
RNA isolation and RT–PCR analysis
RNAwas isolated from frozen cell pellets as previously described17.
Quantitative RT–PCR reactions were performed on an ABI Prism
5700-sequence detection system (PE Applied Biosystems) as detailed
elsewhere18 using specific primers of selected photosynthetic genes
(see Table 1), defined according to their sequences in MIT 9313,
MED4 and SS120 genome data banks. For each quantitative RT–PCR
experiment, measurements were made in duplicate and experiments
were repeated on two distinct cultures.
Electron microscopy
Isolated preparations were placed onto carbon-evaporated, glow-discharged,
300-mesh copper grids and negatively stained with 2% uranyl acetate
and imaged using a Philips CM 100 electron microscope at 80 kV.
The magnification was calibrated to be x50,850. Five electron micrographs
were taken for each preparation where the first minima of the
contrast transfer function was calculated as being in the range
21–22 Angstrom. Micrographs were digitized using a Leafscan
45 densitometer at a step size of 10mm. All image processing was
performed within the IMAGIC-5 software environment19 at a sampling
frequency of 3.92 per pixel on the specimen scale. Single-particle
data sets of approximately 1,200 (Pcb–PSI supercomplex;2Fe,
band 3), 5,900 (áFe, band 2) and 1,600 (2Fe, band 2) were
obtained by selecting all possible single particles from the micrographs.
Reference-free alignment and multi-variate statistical classification20
were used to identify the initial class averages used for iterative
refinement that resulted in the improved class averages shown.
Image processing
Coordinate data sets were obtained from the RCSB Data Bank (http://www.rcsb.org)
under the entry codes for 1JB0 (PSI 2.5 Angstrom resolved structure10)
and 1IZL (PSII 3.7 Angstrom structure11). These structural models
were visualized using the program ViewerLite (Accelrys Inc.) and
overlaid onto the calculated electron-microscopy-derived two-dimensional
projection maps at the same scale. The coordinates of CP43 were
extracted from the 1IZL.pdb file and
modified by the removal of the large loop joining helices Vand VI
so as to better represent a typical Pcb protein. These coordinates
were modelled into the centre of mass assigned to each Pcb subunit.
Received 3 April; accepted 15 July 2003; doi:10.1038/nature01933.
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