the pas - gaf - phy and pas - gaf fragments from deinococcus radiodurans were expressed in the escherichia coli strain bl21 ( de3 ) and purified by affinity and size - exclusion chromatography .
crystallographic data was collected at beamline id23 - 1 of the esrf ( see extended data table 1 ) .
time - resolved x - ray scattering with millisecond time resolution were recorded at beamline csaxs of the swiss light source .
saxs measurements were performed at beamline bm29 of the european synchrotron radiation facility ( esrf ) and analysed as summarised in extended data table 2a .
time - resolved x - ray scattering data in the micro- and millisecond ranges were collected at beamline id-14-b , biocars , of the advanced photon source at argonne national laboratory .
molecular dynamics simulations ( gromacs 4.5.5 ) were used to generate trial solution structures and theoretical scattering curves were evaluated using zernike expansion as implemented in sastbx .
a , statistics of the static saxs ( bm29 ) data , including radii of gyration ( rg ) , maximum particle dimension ( dmax ) , porod volume ( vporod ) , forward scattering ( i0 ) . the molecular weights ( mmexp ) were estimated from the i0 of bsa using the formula m(sample )  m(bsa)*[i0(sample)/i0(bsa ) ] , where m(bsa ) = 66 kda .
a - c , singular value decomposition ( s(q , t ) = u  s  v ) of time - resolved solution scattering data from pas - gaf - phy .
two components suffice to describe the data , the final product ( n=1 ) and a transient low - q depression ( n=2 ) .
a , the first three basis spectra ( 1 , 2 and 3 columns of u.s ) , and original x - ray scattering data ( black ) with reconstruction based on the first two singular values ( all columns of u.s.v red ) are shown .
b , relative amplitudes of the two first basis spectra ( 1st and 2nd columns of v ) .
d , the rise of the pfr product state as measured by direct integration of difference scattering curves ( < s(q ) > 1.2<=q<=2.5 ) and by absorption spectroscopy ( abs . at 754 nm ) .
these data establish that the structural change occurs just after the pfr state is formed in the chromophore .
note the positive signal in the absorption curve with very small amplitude at > 3 ms , which appears to decay while the structural signal rises .
this could be because the absorption properties depend weakly on the large - scale rearrangement .
e , direct static difference data from figure 1 , amplified by q to reveal wide - angle oscillations .
extended data figure 2 | light - induced changes in the secondary structure of the evolutionally conserved phy tongue .
a , secondary structure and topology of the deinococcus radiodurans pas - gaf - phy construct .
the structural elements in our crystal structures are very similar to other published phytochrome structures .
the phy tongue region ( box ) , however , was found to refold upon illumination .
the five - stranded -sheet core of the gaf domain is extended by a small sixth -strand ( called 2 ) that interacts with the phy tongue ( see fig .
the mini - sheet structure at the knot region   is not included in the graph .
b , omit map of the phy tongue in the dark crystal form ( upper panel ) and the illuminated crystal form ( lower panel ) . in the dark crystal form ,
the omit map density ( blue ) supports the built -turn secondary structure ( orange sticks ) , even though most of the side chains are poorly resolved . in the illuminated crystal form ,
the omit map ( blue ) clearly reveals the density of a helix with its bulky side chains ( orange ) .
the omit maps were calculated by repeating molecular replacement and a refinement step ( see supplementary information ) with a structure where the phy tongue was removed .
c , sequence alignment of part of the gaf domain and of phy loop region .
the conserved dip motif in the gaf domain and prxsf motif in the phy tongue are marked by asterisks ( * ) .
five representatives from eubacterial ( bphp ) , cyanobacterial ( cph ) , higher plant , fungi ( fph ) and pas - less phytochromes are shown .
tomato t1 bphp , rhodopseudomonas palustris tie-1 bphp3 , pseudomonas aeruginosa pao1 bphp , agrobacterium fabrum str .
pcc6803 syn - cph1 , microcystis aeruginosa nies-843 , nodularia spumigena ccy9414 , cyanothece sp .
pcc 7822 , anabaena variabilis atcc 29413 , physcomitrella patens phy1 , zea mays phyb1 , populus trichocarpa phya , selaginella martensii phy1 , arabidopsis thaliana phya , synechococcus osb syb - cph1 , synechococcus osa sya - cph1 , nostoc punctiforme pcc73102 , lyngbya sp .
extended data figure 3 | biliverdin structure and spectra in crystals . a , photographs of the crystals under cryogenic conditions at the beamline id23 - 1 .
b , biliverdin omit maps of the dark ( upper left ) and illuminated ( upper right ) form support the existence of the modelled biliverdin conformations ( yellow and orange ) . comparison of the electron density around the biliverdin with published structures ( lower panels ) . in the dark form , the electron density indicates a conformation similar to the published pr structures , including a d. radiodurans structure ( 2o9c , cyan ) .
however , the electron density supports neither the biliverdin as determined in the pfr structure of pabphp ( 3nhq , red ) , nor as determined in the pr structure of d. radiodurans ( 2o9c , cyan ) .
therefore the rotation of the biliverdin d - ring can not be reliably determined and is modelled with both possibilities ( 15za , and 15ea , orange and yellow in upper right panel ) .
omit maps were calculated as in extended data figure 2 and contoured at a sigma level of 3.0 .
c , representative absorption spectra of the dark ( black ) and illuminated ( grey ) crystals , recorded at 123k .
note that the terms  illuminated  and  dark  refer here to the crystallization conditions ( see supplementary information for details ) .
illumination with red light in the crystallization drops at ambient temperature led to a slight increase of far - red absorption and disintegration of the crystals ( data not shown ) .
the spectrum of the illuminated crystals shows that a substantial proportion ( > 50% ) of the proteins reside in pfr state .
the illuminated crystals could be switched to pr - like absorption with far - red illumination .
reversely , also the pfr - like features could be increased with red light ( data not shown ) with illumination at ambient temperature . exposure with light increased the scattering background in the absorption measurements .
the crystals seemed unaffected by the illumination when illuminated with red light in the crystallization drops .
although the spectral analyses of the illuminated crystals do not indicate a pure pfr spectrum , and the biliverdin conformation can not be fit unambiguously to the electron density , the remainder of the electron density is homogeneous ( extended data figure 2b ) .
most importantly , the tongue region of the phy domain adopts the conformation resembling the pfr state of pabphp ( extended data figure 4b ) .
the conformations of the four monomers in an asymmetric unit are practically identical and hence we conclude that biliverdin can co - exist in both pr and pfr states inside this crystal form and still the protein part represents the structural aspects of the pfr state only .
a , comparison of the dark crystal form ( green / dark grey ) to cyanobacterial cph1 in pr state ( pdb code 2vea , orange / light grey ) . 

b , comparison of the illuminated crystal form ( green / dark grey ) to pabphp in pfr state ( pdb code 3nhq , orange / light grey ) . in both the pr and pfr forms ,
key interactions are conserved between the phytochromes ( black dashes ) , as well as the positions of three conserved tongue motives ( see extended data figure 2c ) . the residues of these three motives
/ a)g , prxsf , and ( w / f , y)xe , with numbering from the d. radiodurans sequence .
trp451 was not modelled in our illuminated crystal structure , and part of the phy tongue has been removed for clarity .
small changes in relative orientations between the difference crystal structures are observed , e.g. a slight tilt of helix of the pfr tongue .
a , experimental saxs data of dark (  pr  ) and pre - illuminated (  pfr  ) samples .
the data is merged from the concentration series ( extended data table 2b ) and normalized on 0.4 nm < q < 0.6 nm .
b , guinier plot of the low - q region , shown for all concentrations .
inset shows the radii of gyration ( rg ) calculated from the curves in ( a ) according to the guinier approximation .
c , average difference scattering signals calculated from the solution - structural models using three methods : crysol ( default settings ) , sastbx with spherical harmonic expansion ( she , default settings ) , and sastbx with zernike polynomial expansion as described in supplementary information .   the choice of calculation method does not significantly change the predicted x - ray difference scattering .
d , determination of the relative pr / pfr populations represented by the bm29 data as described in supplementary information .
we find that our pr sample contained only pr ( top ) whereas in the pfr sample 64% of the protein molecules adopted the pfr conformation ( bottom ) .
1 ) represent , up to a scaling factor , the relation between pure pr and pfr populations .
this is in contrast to traditional saxs which report on population mixtures , because the pfr state can not be easily produced with 100% population in solution .
crystal packing interactions of the ( a ) dark and ( b ) illuminated crystal forms .
the dimer of an asymmetric unit is shown in red and the symmetry mates in grey .
, crystal contacts are seen in the top regions of the phy domains and therefore may cause artefacts in the long scaffolding helix and in the opening of the phy domains . in the illuminated form contacts are such that the phy domains may be pushed closer together ,
which is consistent with the larger separation of the phy domains as refined from the solution x - ray scattering data .
it is noteworthy that the relative orientation of the monomers in the dimer is different between all three known structures for pas - gaf - phy phytochromes . for pseudomonas
aeruginosa the dimer is parallel with variations in between different copies of the dimer in the crystallographic unit cell , in two cyanobacterial phytochrome an antiparallel dimer is observed , and in our pr structure , the monomer have an angle of approximately 45. extended data figure 7 | solution - structural refinement .
a , the distribution of phy domain separations ( rpp ) obtained from unbiased md simulations ( production runs 1 - 3 ) .
3 are indicated by crosses ( with n = 100 , m  610 ) .
while the pr structures cluster in a region of high sampling , the pfr structures lie at the edges of the phy - phy distribution , suggesting inadequate sampling . to remedy this
, we artificially scanned the phy domain separation in separate simulations ( production runs 4 and 5 ) to improve sampling .
b , the new distribution of pulled phy domain separations in the pfr state . the final analysis and all solution - structural conclusions drawn in this study
are based on the trajectories described in b. c - e , consistency test of the structural refinement procedure .
c , a cutoff parameter rcut was introduced to reject all md frames rpp < rcut .
the resulting average over rpp of the best n = 100 pairs is plotted as a function of rcut .
it is found that rpp  rcut , which indicates that the best fit to the difference x - ray scattering data is always at the highest separations available in sampling range .
d and e show the dependence of the total and average error as a function of rcut , respectively . it is observed that the error decreases steeply for rcut  5 nm , and only marginally forrcut  5 nm .
we therefore consider optimization in the latter range overfitting , and applied rcut = 5.0 nm in the refinement for the solution structures .
 sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains . 
 phytochromes are a major family of red - light sensing kinases that control diverse cellular functions in plants , bacteria , and fungi.1 - 9 bacterial phytochromes consist of a photosensory core and a c - terminal regulatory domain.10,11 structures of photosensory cores are reported in the resting state12 - 18 and conformational responses to light activation have been proposed in the vicinity of the chromophore.19 - 23 however , the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive . here , we report crystal and solution structures of the resting and active states of the photosensory core of the bacteriophytochrome from deinococcus radiodurans . the structures reveal an open and closed form of the dimeric protein for the signalling and resting state , respectively . 
 this nanometre scale rearrangement is controlled by refolding of an evolutionarily conserved  tongue  , which is in contact with the chromophore . 
 the findings reveal an unusual mechanism where atomic scale conformational changes around the chromophore are first amplified into an ngstrm scale distance change in the tongue , and further grow into a nanometre scale conformational signal . 
 the structural mechanism is a blueprint for understanding how the sensor proteins connect to the cellular signalling network . 
METHODS SUMMARY
Supplementary Material