purification of squid rhodopsin  all procedures were carried out at room temperature in the dark or under dim red light unless otherwise indicated . squid rhodopsin was prepared from todarodes pacificus caught in the japan sea , using previously described methods ( 13 ) . the membranes were treated with v8 protease ( pierce , 50:1 w / w of rhodopsin : v8 protease ) at room temperature for 1 h to remove the unique c - terminal proline - rich extension of the squid rhodopsin . the reaction was terminated by extensive washing with hepes buffer ( 5 mm hepes , ph 7.0 , 1 mm edta , 1 mm dithiothreitol ) . the membranes were solubilized with 2% ( w / v ) dodecyl maltoside ( ddm , anatrace ) for 1 h at 4 c . after centrifugation , the supernatant was loaded onto a deae - cellulose column ( whatman ) equilibrated with buffer a ( 50 mm hepes , ph 7.0 , 0.05% ( w / v ) ddm ) . the unbound fraction was collected and applied to a concanavalin a - sepharose 4b column ( amersham biosciences ) equilibrated with buffer a. the rhodopsin was eluted with 0.2 m -methyl mannoside solution . fractions containing squid rhodopsin were pooled and dialyzed against buffer a and then concentrated by ultrafiltration ( amicon ultra , millipore ) . n - terminal sequencing and mass analyses  the identity and integrity of the purified protein were assessed by n - terminal amino acid sequencing by edman degradation and various mass spectrometric analyses , including maldi - tof / ms , maldi - tof / tof - ms / ms , nano - liquid chromatographyquadrapole tof - ms / ms , and high performance liquid chromatography - electrospray ionization - iontrap - ms / ms as described in the supplemental materials ( 14 ) . one microliter of protein sample ( 10 mg / ml ) in a solution of 10 mm hepes , ph 7.0 , 200 mm nacl , 2 mm dodecyldimethylamine oxide , 0.03% ( w / v ) ddm was mixed with 1 l of reservoir solution ( 0.1 m hepes , ph 7.0 , 8% ( v / v ) ethylene glycol , 28% ( w / v ) polyethylene glycol 400 ) and left to equilibrate at 20 c . structure determination and refinement  x - ray diffraction data were collected at 100 k on beam line bl45xu at spring-8 . the structure was determined by molecular replacement with the program molrep in the ccp4 program suite ( 16 ) using a monomer of the trigonal crystal structure of the bovine rhodopsin ( pdb code : 1gzm ) ( 17 ) as a search model . refinement and model building were performed iteratively with the programs cns ( 18 ) , refmac5 in ccp4 ( 16 ) , and o ( 19 ) . during refinement , we used grouped , unrestrained b - factor refinement with a single group for the entire molecule . all refinements were carried out with 10% of the reflections for cross validation . despite the low resolution data and the low sequence homology between squid and bovine rhodopsins ( 24% ) , the structure was well refined thanks to the structural similarity of the transmembrane helices and the positions for a disulfide bridge , an 11-cis - retinal chromophore in the schiff base linkage , and the conserved residues . b - factor sharpening was used to generate detailed maps using the cns program with bsharp values ranging from 50 to 150  ( 20 ) . data collection and refinement statistics are shown in table 1 . all figures including electrostatic potential surfaces were prepared using pymol ( delano scientific llc ) . the coordinates have been deposited in the protein data bank ( pdb ) with the accession code 2ziy . table 1data collection and refinement statisticsdata collection  wavelength (  )   0.97950   resolution (  )   43.2 - 3.7   measured reflections   43,571   unique reflections   6,680   completeness ( % ) a 93.3 ( 74.3 )   rmerge ( % ) b 6.4 ( 77.5)c  space group c2221  unit cell (  ) a = 84.3 , b = 108.7 , c = 142.2 refinement  resolution (  )   43.2 - 3.7   reflections used   6,647   rwork / rfree ( % ) de 30.2/33.0 ( 41.4/43.2 )   r.m.s.f deviation   bond (  )   0.014   angle (  )   2.01   ramachandran statistics   most favored region ( % )   70.4   additional allowed region ( % )   27.1   generously allowed region ( % )   2.1   disallowed region ( % )   0.3 avalues in parentheses are for the highest - resolution shell ( 3.83 - 3.70 ).brmerge = i|i(h)i  i(h)|/i|i(h)i| , where i(h) is the mean intensity of equivalent reflections.cthe last shell rmerge is rather high as a result of strong anisotropy.drwork = |fo  fc|/|fo| , where fo and fc are the observed and calculated structure factor amplitudes , respectively.erfree = |fo  fc|/|fo| , calculated using a test data set , 10% of total data randomly selected from the observed reflections.fr.m.s . , root mean square .   data collection and refinement statistics values in parentheses are for the highest - resolution shell ( 3.83 - 3.70  ) . rmerge = i|i(h)i  i(h)|/i|i(h)i| , where i(h) is the mean intensity of equivalent reflections . rwork = |fo  fc|/|fo| , where fo and fc are the observed and calculated structure factor amplitudes , respectively . rfree = |fo  fc|/|fo| , calculated using a test data set , 10% of total data randomly selected from the observed reflections . chemical structure of squid rhodopsin  we first confirmed the chemical structure of v8 protease - treated squid rhodopsin . n - terminal sequencing and various ms analyses showed that the squid rhodopsin was cleaved at glu-373 by the v8 protease , and the polypeptide chain was from gly-2 to glu-373 containing a val to ile substitution at position 18 with all the expected post - translational modifications . these included n - glycosylation and two palmitoylations , as well as a schiff - based retinal . the molecular mass of squid rhodopsin was 43,842 da ( calculated molecular mass 43,883 ) by maldi - tof ms analysis ( supplemental fig . 1 ) ( 14 , 21 , 22 ) . overall structure  the overall structure of c - terminal truncated squid rhodopsin is a class a gpcr , with seven transmembrane helices , including the amphipathic short helix h8 and a prominent , well ordered cytoplasmic region ( see fig . all amino acid residues except the first 2 residues were defined in the structure , with a disulfide bridge between cys-108 and cys-186 in the extracellular region , the 11-cis - retinal connected to lys-305 by the schiff base , and the two palmitoyl thioesters of cys-336 and cys-337 . we did not model the n - glycan of asn-8 in the structure due to vague maps , although there were substantial electron densities in 2fo  fc and fo  fc maps where the glycan would be located ( supplemental fig . squid rhodopsin has seven transmembrane helices ( th1th7 ) connected by three extracellular ( el1el3 ) and three cytoplasmic ( cl1cl3 ) loops with n- and c - terminal tails on the extracellular and cytoplasmic sides of the membrane ( fig . the transmembrane helices bend around the conserved proline or glycine residues , especially those with adjacent bulky aromatic residues ( fig . 1 ) ( 5 , 8) . the amphipathic h8 is located just after th7 at the putative cytoplasmic surface of the membrane and is a conserved structure in rhodopsin - like gpcrs ( 5 , 8 , 23 ) . the transmembrane helices of squid rhodopsin can be superimposed on those of bovine rhodopsin and human 2ar with the root mean square deviations of 1.5 and 2.2  , respectively , indicating that the transmembrane helical architecture of the three gpcrs are well conserved , despite many bends in the helices .   figure 1.structural sequence alignment of squid rhodopsin , bovine rhodopsin and 2ar . residues on helix regions are colored red , and residue(s ) of helix bending are colored in blue . transmembrane helical regions ( th1th7 ) and helix h8 with extracellular ( el1el3 ) and cytoplasmic ( cl1cl3 ) loops , and each helix in n- and c - terminal tails ( nh and ch ) are indicated . posttranslational modifications are shaded by the following colors : cyan , n - glycosylation ; a pair of pink or green , disulfide bridge(s ) ; yellow , palmitoylated cysteine ; blue , schiff - based lysine with 11-cis - retinal ; gold , n - terminal methionine acetylation . residues indicated by small letters are not in models but in crystal protein samples , and residues indicated by small letters in italic gray do not exist in the crystal sample proteins due to expression processing , protease digestion , or protein engineering . squ_rhod , squid rhodopsin ( pdb code : 2ziy in this study ) ; bov_rhod , bovine rhodopsin ( 1f88 ( 5 ) or 1gzm ( 17 ) ) ; and adrb2 ( 2rh1 ( 8) ) .   structural sequence alignment of squid rhodopsin , bovine rhodopsin and 2ar . residues on helix regions are colored red , and residue(s ) of helix bending are colored in blue . transmembrane helical regions ( th1th7 ) and helix h8 with extracellular ( el1el3 ) and cytoplasmic ( cl1cl3 ) loops , and each helix in n- and c - terminal tails ( nh and ch ) are indicated . posttranslational modifications are shaded by the following colors : cyan , n - glycosylation ; a pair of pink or green , disulfide bridge(s ) ; yellow , palmitoylated cysteine ; blue , schiff - based lysine with 11-cis - retinal ; gold , n - terminal methionine acetylation . residues indicated by small letters are not in models but in crystal protein samples , and residues indicated by small letters in italic gray do not exist in the crystal sample proteins due to expression processing , protease digestion , or protein engineering . squ_rhod , squid rhodopsin ( pdb code : 2ziy in this study ) ; bov_rhod , bovine rhodopsin ( 1f88 ( 5 ) or 1gzm ( 17 ) ) ; and adrb2 ( 2rh1 ( 8) ) . transmembrane helices are indicated as th1th7 , and amphipathic short helix h8 is indicated as h8 . 11-cis - retinal at lys-305 and palmitoylated cysteines cys-336 and cys-337 are indicated by the yellow sphere - and - stick models . a hydrophilic short helix in each n- and c - terminal tail is indicated as nh and ch , respectively . n and c termini are indicated by the letters n and c , and the cytoplasmic loop 3 is indicated as cl3 . squid rhodopsin in this study is shown in orange , bovine rhodopsin in the trigonal crystal ( 1gzm ) ( 17 ) is blue , and 2ar ( 2rh1 ) excluding the t4 lysozyme part of cl3 ( 8) is sky blue . the electrostatic surfaces of squid rhodopsin ( squ rhod ) , bovine rhodopsin in the trigonal crystal ( bov rhod ) , and 2ar ( 2ar ) are represented in blue ( positive ) to red ( negative ) with the squid rhodopsin structure in an orange schematic . to clarify the contribution of the distal c - terminal tail , the electrostatic surface of squid rhodopsin without the c - terminal tail after glu-343 transmembrane helices are indicated as th1th7 , and amphipathic short helix h8 is indicated as h8 . 11-cis - retinal at lys-305 and palmitoylated cysteines cys-336 and cys-337 are indicated by the yellow sphere - and - stick models . a hydrophilic short helix in each n- and c - terminal tail is indicated as nh and ch , respectively . n and c termini are indicated by the letters n and c , and the cytoplasmic loop 3 is indicated as cl3 . b , superimposed schematic models and electrostatic surfaces of known gpcr structures . superimposed schematic structures of squid and bovine rhodopsins , and 2ar are shown . squid rhodopsin in this study is shown in orange , bovine rhodopsin in the trigonal crystal ( 1gzm ) ( 17 ) is blue , and 2ar ( 2rh1 ) excluding the t4 lysozyme part of cl3 ( 8) is sky blue . the electrostatic surfaces of squid rhodopsin ( squ rhod ) , bovine rhodopsin in the trigonal crystal ( bov rhod ) , and 2ar ( 2ar ) are represented in blue ( positive ) to red ( negative ) with the squid rhodopsin structure in an orange schematic . to clarify the contribution of the distal c - terminal tail , the electrostatic surface of squid rhodopsin without the c - terminal tail after glu-343 different th5 regions are indicated by the red line . the conformation of the n- and c - terminal tails and the cl2 , cl3 , and el3 loops differs significantly between bovine and squid rhodopsins , whereas the el2 conformation is similar to bovine rhodopsin to accommodate the same 11-cis - retinal ligand found in visual chromophores . in the proximal c - terminal tail , squid rhodopsin has a 5-residue insertion between h8 and the palmitoylated cysteines , whereas bovine rhodopsin and 2ar have a palmitoylated cysteine immediately after h8 ( fig . 1 ) ( 6 , 8) . in the large helix bundle cavity on the extracellular side , 11-cis - retinal bonds to lys-305 with a putative protonated schiff base , which is countered by the hydroxyl group of tyr-111 as well as sandwiched by the amides of asn-87 and asn-185 at both sides of the retinal within hydrogen bond distances from the schiff base nitrogen atom ( 21 ) . the architecture surrounding the schiff base is consistent with a smaller blue shift upon photoactivation than that of bovine rhodopsin , whose schiff base is directly countered by the charged glu-113 ( 5 , 24 ) . the extended th5 and th6 and the cytoplasmic loops fold into the compact structure ( fig . 2 ) , although squid rhodopsin has longer helices than bovine rhodopsin and 2ar on the cytoplasmic side ( 5 , 8) . squid rhodopsin has a 12-amino - acid insertion in the cl3 region when compared with bovine rhodopsin ( fig . th5 elongates to lys-239 with a bend at his-230 located at the putative cytoplasmic surface of the membrane , in addition to a small bend at gly-208 . th6 extends from glu-245 with a bend at ser-275 before pro-276 and tyr-277 ( fig . the residues from asp-341 to asp-347 form an additional short hydrophilic c - terminal helix ch . after helix ch , the c - terminal tail from lys-348 to glu-373 ( which is the c terminus in the v8-treated sample ) interacts closely with the extended th5/th6 region . the c - terminal region returns to the putative membrane surface in an extended structure via polar interactions with cl2 ( fig . , the truncated residual polyproline region may align along the surface of the plasma membrane to form the ordered rhabdomeric structure in squid photoreceptor cells ( 21 ) . interaction with g - protein  the cytoplasmic region is folded compactly in squid rhodopsin as described and is thought to interact with the coupling of gq . the folded region consists of cl2 , the helix bundle end of th5 and th6 extruded into the cytoplasm as the extended cl3 region , and h8 as well as the distal c - terminal tail including the ch helix . except for the distal c - terminal tail , these regions interact directly with the respective g - protein in bovine rhodopsin , as shown by peptide competition studies ( 32 ) . the electrostatic potentials of the cytoplasmic surfaces have a profile characteristic of intracellular gpcr domains ( fig . the distinct electrostatic profiles between these structures are located around the th5 intracellular surface region of the putative plasma membrane . interestingly , the corresponding th5 region was important to gi coupling but less so to gq in blt1 ( 28 ) . chemical cross - linking studies of the active form of bovine rhodopsin showed that the cl3 center residues are located close to the gt - coupled site ( 29 , 30 ) . in the two different crystal forms of bovine rhodopsin , cl3 showed prominent , different conformations ( 5 , 6 , 17 ) , whereas the cl3 region was disordered or substituted in both 2ar structures ( 7 , 8) . in the trigonal bovine rhodopsin crystal , cl3 adopts an extended helix - like conformation similar to that of squid rhodopsin ( fig . 2b ) ( 17 ) but not in the tetragonal crystal due to hindrance in the crystal packing by contact with neighboring molecules ( 5 , 6 ) . the compact hydrophilic intracellular domain of squid rhodopsin on the plasma membrane should participate in recognizing the g - protein during activation . like other class a gpcrs ( 26 , 27 ) , squid rhodopsin with h8 anchored by the cysteine palmitoylations ( fig . 2a ) effectively encounters the g - protein anchored by lipid modification(s ) on the lateral membrane surface ( 25 ) . however , the mechanism of g - protein activation by gpcr remains to be further elucidated .
g - protein - coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via g - proteins . 
 rhodopsin is a prototypical g - protein - coupled receptor involved in the retinal visual signaling cascade . 
 we determined the structure of squid rhodopsin at 3.7 resolution , which transduces signals through the gq protein to the phosphoinositol cascade . 
 the structure showed seven transmembrane helices and an amphipathic helix h8 has similar geometry to structures from bovine rhodopsin , coupling to gt , and human2-adrenergic receptor , coupling to gs . 
 notably , squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with g - proteins , and this region is flexible or disordered in bovine rhodopsin and human2-adrenergic receptor . 
 the transmembrane helices 5 and 6 are longer and extrude into the cytoplasm . 
 the distal c - terminal tail contains a short hydrophilic -helix ch after the palmitoylated cysteine residues . 
 the residues in the distal c - terminal tail interact with the neighboring residues in the second cytoplasmic loop , the extruded transmembrane helices 5 and 6 , and the short helix h8 . additionally , the tyr-111 , asn-87 , and asn-185 residues are located within hydrogen - bonding distances from the nitrogen atom of the schiff base .