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Data acquisition and processing. Small (80 μm by 80 μm by 150 μm) crystals were excited using single 4-mJ laser pulses (7 ns FWHM, ≤1.5-mm-diameter beam cross section at the crystal, 495-nm wavelength) from a coumarin 500-dye laser (Continuum) pumped by the third harmonic of a Nd:YAG laser (Continuum) timed to arrive at specified times (between 1 ns and 1 ms) in advance of a single x-ray pulse or pulse train. This delay is defined as the time from the peak of the laser pulse to the edge of the x-ray pulse (14). A 70-pole wiggler and a hybrid 46-pole undulator (wavelength range of 0.3 to 1.8 Å) were used simultaneously to further enhance the x-ray intensity at the crystal. Single x-ray pulses were isolated with a microsecond shutter train (14) from the ESRF storage ring operating in single-bunch mode at a machine current of 15 mA. Laue diffraction intensities were collected on an image-intensified charge-coupled device detector (Thompson; ESRF). To minimize systematic errors such as those related to x-ray absorption, we collected a dark data set for each crystal in addition to the photoexcited data sets. Data sets consist of 30 images taken at crystal angular settings separated by 2° through a range of 60°. Each image is a composite of 10 150-ps exposures accumulated on the detector before readout, to enhance the relatively weak diffraction data collected in single-bunch mode. No significant radiation damage was detected over the course of data collection. Data were reduced and scaled with the LaueView software package (28). Laue reflections stimulated by several energies (multiples) were successfully deconvoluted and included in the final data set, which improved the completeness at low resolution and the overall redundancy.
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unpublished results
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Extent of photoinitiation. Optical thickness directly affects the extent of photoexcitation in the crystal. Crystals that are too thick will have a lower extent of photoinitiation and a highly nonuniform excitation throughout their volume (9). With 7-ns laser pulses at longer wavelengths, the possibility of photoexcitation of molecules in the [pR] state is encountered, which may stimulate reisomerization of the [pR] state back to pG. We demonstrated (V. Šrajer and B. Perman, unpublished results) that the photocycle can be successfully initiated in the crystal under our experimental conditions, and estimate that 12% of the molecules are converted to the [pB] state.
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Šrajer, V.1
Perman, B.2
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7144260741
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personal communication
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free. Coordinates for the [pR] state of PYP have been deposited in the Brookhaven Protein Data Bank under accession code 2pyr.
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Zhou, F.1
Bash, P.2
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7144252224
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note
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We thank R. Cordfunke for expert assistance in the preparation of PYP samples, M. Roth for use of the pulsed laser, S. Labouré for laser technical assistance, and W. Schildkamp for participation in time-resolved x-ray crystallographic technique development. K.M. thanks D. McRee and E. Getzoff for introducing him to PYP. Supported by grants from NIH to K.M.
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