fig1 a is a listing of the three - dimensional co - ordinators of the atoms of the α and β subunits of hcg . the atom listing is preceded by a cryst1 card which gives the dimensions ( in å ) of the crystallographic unit cell . the next three cards define a matrix which converts co - ordinates from orthogonal angstrom to fractions of the unit cell . each atom card gives the ( arbitrary ) atom number , the atom label as per the attached sheet ( the atoms of each amino acid main chain are labelled n . ca , c , o ; the atom type is inferred from the first letter of the label and the atom position α , β , δ , ε , ζ , η , is given as a , b , g , d , e , z , h respectively ), the amino acid residue type , the protein chain label ( a for α ; b for β ) and the amino acid sequence numbers ( for the β - subunit , 100 has been added to each number ). the first three numbers give the orthogonal x , y , z co - ordinates of the atom in å . the next number is an occupancy number and would be less than 1 . 0 if the atom could be seen in more than one position i . e . the amino acid could be seen in more than one orientation . the final number is a temperature factor which relates to the thermal amplitude of vibration of the atom . the atom numbering is arbitrary and the calculated coordinates of h atoms are omitted . at the end of the listing are carbohydrate residues labelled nag ( for n - acetylglucosamine ). these are attached to the protein at four positions ( asn 52 and 78 on α , asn 13 and 30 on β ). conditions for crystallising hf treated hcg ( hf - hcg ) have been reported ( harris at al ., 1989 ) and it is these crystals which have been used in the structure determination reported here . hf treated hcg crystallises from ammonium sulphate solutions ( harris et al ., 1989 ) as hexagonal bipyramids with the space group p6 522 and cell dimensions a = 88 . 68 , c = 177 . 24 å . the crystals diffract to 3 . 5 å resolution on a laboratory x - ray source and to 2 . 5 å with synchrotron radiation , though they are extremely sensitive to radiation damage in the synchrotron beam . the structure has been determined to 3 . 0 å resolution by multiple isomorphous replacement ( mir ) with relatively poor heavy atom derivatives . solvent flattening made a marked improvement to the phasing , but not sufficient to allow an unambiguous tracing of the electron density map , or a determination of the boundary between the α / β subunits . using the program mice rounds of maximum entropy ( me ) solvent flattening were performed with one round to phase permutation . further rounds of me solvent flattening , in combination with partial structure phase combination , produced significant improvement in the phasing . the final electron density map allows a tracing of the α - subunit from residues 5 to 89 and the β - subunit from 3 to 112 . the residue β - 112 is open to a large solvent cavity and we assume that the remaining c - terminal 33 residues adopt a random conformation and are thus not visible in the map . the initial model was refined using the program xplor . five rounds of refinement and model building gave a final structure . no solvent molecules but six n - acetylglucosamine molecules positioned in good density have been included . a geometric analysis of the refined structure with the program procheck gave values better than expected for structures determined at this resolution . central to the successful map interpretation and thus the three - dimensional structure elucidation was the recognition of the disulphide bridges and their correct assignment to positions in the amino acid sequence for each subunit . this assignment is different from the one generally accepted in the literature . there are five disulphide bonds in the α - subunit and six in the β - subunit . the chemical assignment of the disulphide pairings has been the topic of many studies ( for review see ryan et al ., 1987 ), with no complete agreement having been reached . however , it has been accepted that the following assignments are correct : α7 - 31 , α10 - 32 , β93 - 100 , β26 - 110 and perhaps β23 - 72 . from the me phased map some electron density could be interpreted as possible disulphide connections , but the close proximity of these regions prevented any unambiguous interpretation . however , some continuous lengths of density could be fitted with a polyalanine chain . in one of these , the separation between the positions of possible disulphide links positioned cys 23 , cys 26 and cys 34 of the β - subunit . assuming the s -- s linkages given by prior workers ( 23 - 72 , 26 - 110 , 34 - 88 ) enabled a sequence to be fitted from β3 to β38 , β60 to β89 and β93 to β112 with the disulphide bridge β93 - 100 formed . with this partial sequence fitted , it became apparent that the remaining s -- s bridges could not be formed as expected ( i . e . 38 - 57 , 9 - 90 ). the map could be interpreted only if the remaining linkages were formed from 9 to 57 and 38 to 90 . this combination produces an unusual knot of three disulphide bridges . once density had been assigned to the β - subunit , the remaining density contained a feature which could be recognised as a similar knot of cystines , but in order to fit the amino acid sequence to the density , disulphide linkages ( 10 - 60 , 28 - 82 , 32 - 84 ) different to those previously assumed were necessary . from this core , the remainder of the α - subunit could be traced . this cystine - knot motif involves three disulphide bridges arranged such that two disulphides link adjacent anti - parallel strands of the peptide chain and form a ring through which the third disulphide penetrates . as well as a common arrangement in the sequence order of half cystines , two amino acid sequence motifs are associated with the cystine knot . both of these sequence patterns are found in the subunits of hcg ; α28 - 32 ( cmgcc ), α82 - 84 ( chc ), β34 - 38 ( cagyc ) and β88 - 90 ( cqc ). the disulphide pairings are given in table 2 . of the linkages not predicted in any previous study , ( α10 - 60 , 28 - 82 , 32 - 84 , 59 - 87 ; β9 - 57 , 38 - 90 ) all but one are involved in the cystine knots . there is an unusual bend at asn 15 of the α - subunit , where the sidechain of asn forms hydrogen bonds to the main chain nh of residues 17 and 18 resulting in pro 16 . phe 17 and phe 18 bulging away from the antiparallel strand and forming a prominent feature on the surface of the molecule . the subunits each have a similar fold which is determined largely by the central cystine knot . on one side of the knot , there is a loop of double - stranded β - sheet like structure , while on the other side there are two hairpin loops lying in almost parallel plates . in the β - subunit , these hairpins are linked by the cys23 - 72 disulphide bond . fig6 a and 6b show the fold of each subunit and indicate the positions of the disulphide bridges in each . the hairpin structure of each subunit is stabilised by a hydrophobic core extending between the two loops . the α - subunit core consists of phe α17 , phe α18 , ile α25 , val α68 , val α70 , met α71 , phe α74 and val α76 , with the three phenylalanines clustered together and partially exposed to the surface . the β - subunit core includes the residues leu β16 , val β18 , ile β27 , val β29 , ile β67 , leu β69 , val β80 and tyr β82 which buries the disulpuhide bridge β23 - 72 to some extent . in the c - terminal hairpin loops of each subunit there is a pair of β - bulges occurring at adjacent positions in each strand . in the β - subunit , both of these bulges have a ` classical ` hydrogen - bonding pattern . in the α - subunit the hydrogen bond formation is less complete . the structures of all growth factors containing a cystine - knot have a β - bulge which is always located immediately before cys ( v ) in the cystine knot , suggesting that it exists because of the arrangement of the cystine knot . without the bulge , the normal right - handed twist always found in extended loops would lead to a steric clash between side chains on the inner side of the loop and the cys ( ii )- cys ( v ) disulphide in the cystine knot . the other hairpin loop has no equivalent feature as here the twist of the strands takes the side chain away from the disulphide . in the isolated subunits the disulphides within the cystine knots show varying degrees of solvent accessibility . the disulphide cys ( i )-( iv ) is buried in the knot , cys ( ii )- cys ( v ) is buried due to the contribution of three flanking residues ( in a leu 12 , ala 62 and his 79 ; in β tyr 59 , ala 85 and pro 111 ) but cys ( iii )- cys ( vi ) is left exposed to the solvent as are the retaining disulphide bridges . in the α - subunit , the loop on the other side of the cystine knot consists of a stretch of anti - parallel β - sheet made up from residues 35 to 39 and 52 to 56 . the three residues pro 38 - thr 39 - pro 40 , which are conserved in all 18 α - sequences determined , break the β - sheet and lead into two turns of a 3 10 - helix involving residues 40 to 46 . this is the only helical structure in either subunit . the combination of a pro - thr - pro sequence followed by a helix results in the end loop bending away from the β - sheet at an angle of about 100 ° to form a quite rigid structural element which is further stabilised by a h - bond between the thr 39 side - chain and the main - chain on the anti - parallel strand . this conformation places asn 52 on a bend which directs the n - linked carbohydrate on this residue away from the α - chain . at the extreme end of this loop are two conserved hydrophobic residues . leu 48 and val 49 , which are both fully exposed to the solvent . in contrast , the corresponding region of the β - subunit is less constrained and forms a very open loop . away from the cystine knot region , there are no main chain h - bonds between the anti - parallel strands . instead , the loop is stabilised by two side chain to main chain interactions . the side chain of arg 43 is h - bonded to main chain carbonyls of pro 50 and n of leu 52 and the side chain of gln 54 is h - bonded to the main chain n of met 41 . one side of the loop comprising residues 38 to 45 , is held by association with the α - subunit . residues 46 to 49 at the top of the loop are poorly defined in the electron - density map . in the crystal a two - fold axis of symmetry causes residues 48 - 50 to pack against the same residues from a neighbouring molecule . with the exception of the cystine - knot motif no sequence homology has been detected between the two subunits , but when residues forming the cystine knot in each protein are superimposed , a remarkable similarity in structure is apparent ( fig7 ). the cα positions of 49 residues can be superimposed with a rms difference of 1 . 9 å . the two structures have a common core structure and differ mainly in the extent of the loop regions . in the β - subunit the pair of hairpin loops are extended in size , while the double stranded loop on the other side of the cystine knot is longer in the α - subunit . fig5 c and 6c show the arrangement of the heterodimer . the two subunits are arranged with an approximate two - fold symmetry relating one subunit to the other . the segments of well defined β - sheet structure near the cystine knot in each subunit are brought together to form a short seven - stranded β - barrel . one face of this barrel consists of a short strand from the β - subunit ( 97 - 101 ) forming a parallel β - sheet with ( 52 - 60 ) which is part of a four stranded anti - parallel β - sheet consisting of α ( 52 - 60 ), α ( 37 - 29 ), β ( 33 - 40 ) and β ( 12 - 9 ). ( the order of the sequence numbers indicates the direction of the strand ). the central anti - parallel strands formed between the subunits includes the β34 - 36 ( cagy sequence ) which has long been implicated in the formation of the dimer . the other face of the barrel is made up of two anti - parallel strands ( 84 - 90 , 63 - 54 ) of the β - subunit . this complete association buries a surface area between the subunits . away from this central region , there are hydrophobic contacts between val 44 and leu 45 of β and the triplet of phe residues ( 17 , 18 , 74 ) in the α hairpin loop . an unexpected feature of the dimer association is the role played by the loop between cys 93 and cys 110 in the β - subunit . this loop is an integral part of the β - subunit structure as disulphide bridges are formed between cys 93 - 100 and cys 26 - 110 . ( fig6 b ). however , in the heterodimer this loop is wrapped over the α - subunit while remaining covalently bonded to the β - subunit through the disulphide linkages , giving the loop the appearance of a seat belt . there is close association between the inner surface of the belt and the α - subunit . a short strand of parallel β - sheet between β ( 97 - 101 ) and α ( 53 - 57 ) forms part of the central β - barrel . the side chains of the α - subunit residues covered by the belt point towards the belt and interact with either main chain or side chain atoms . the side chain of α35 arg is h - bonded to the side chain of β106 his and to the main chain carbonyl of β104 lys . non - bonded interactions are formed between α37 tyr , β107 pro and β108 leu and between α54 thr and β99 asp . the side chain of α56 glu is h - bonded to the main chain of β104 lys . there are no contacts which are specific to the hcg molecule which would prevent this seat - belt arrangement existing in all of the glycoprotein hormones . the presence of cav at asn52 on the α - subunit would make it impossible for the α - subunit to slide under the β belt in the formation of the heterodimer and the most likely mechanism is one where the β26 - 110 disulphide bond is not fully formed until the dimer association is complete . experiments performed by prior workers have shown that the formation of this disulphide is not completed until after the α / β association has occurred . in its native form hcg is heavily glycoslylated with carbohydrate making up approximately 34 % by weight of the protein . there are two n - linked carbohydrates occuring at asn 52 and asn 78 on the α - subunit and two at asn 13 and asn 30 on the β - subunit . in addition the β - subunit has four o - linked carbohydrates at ser 121 , 127 , 132 and 138 . treatment of the protein with anhydrous hf removes much of the carbohydrate . the hf treatment appears to leave the o - linked carbohydrate largely intact and will truncate the asn linked carbohydrate on the α - subunit to asn -( glcnac ) 2 ( man ) 3 . there is clear electron density for some of the n - linked complex carbohydrates of both subunits . the o - linked residues are not seen . in α , the carbohydrates are found at differing extremities of the molecule . asn 52 is associated with the double stranded loop and asn 78 is near the end of the twisted β - hairpins . the carbohydrate makes few hydrogen bonds to the protein but the first glucnac sugars is in close nonbonded contact with the protein and therefore could be expected to have an influence of the protein conformation . in the β - subunit carbohydrates both n - linked are located together with the cα &# 39 ; s of asn 13 and asn 30 only 7 å apart . this may explain why a number of glycoprotein hormones can have one or other of these asn residues substituted without loss of function . in the hormones with both glycosylation sites , we would expect significant interactions between both carbohydrate chains . the sugars in the β - subunit , in contrast to those on the α - subunit , are exposed on the outward facing β strand of the first β - hairpin and form neither hydrogen bonds nor significant nonbonded contacts to the protein . the overall distribution of the carbohydrate in the heterodimer is in two regions , the first ( asn 78 ) is between the twisted β - hairpins of α , while the second is a central band formed from the other n - linked carbohydrate , which spreads over both faces of the β - subunit ( fig3 ). the only carbohydrate which makes interactions at the subunit interface is at α52 asn which makes contacts to the β - subunit residues , tyr 59 , val 62 , phe 64 , and ala 83 as well as thr 97 , from the determinant loop . hcg differs from hlh and the other glycoprotein hormones in having an extended amino acid sequence at the c - terminal end . this 31 residue c - terminal peptide has an unusual amino acid sequence , containing nine pro and eight ser residues , four of which are glycosylated . although there is space in the crystal lattice to accommodate this peptide , the electron density after asp 112 is very weak and it has not been possible to model any further residues for this portion of the structure . the most likely explanation is that the peptide does not adopt an ordered conformation and is thus highly mobile in the solvent cavity . it is also conceivable that the chain is lost due to acid catalysed cleavage of the peptide within the crystal but the observation that crystals of asialo - hcg are isomorphous with hf - hcg suggests the peptide is disordered in the crystal . a number of residues important for the activity of hcg have been identified through chemical modification of the whole protein or subunits , or through the use of synthetic peptides in competitive inhibition studies . also with the availability of recombinant hcg site - directed mutagenenis has been used . the present model further rationalises these findings and in particular the three - dimensional structure may be used in a test system for drug discovery . the combined results from all these approaches has been to locate a number of regions in both the α and β - subunits which contribute to receptor binding , ( see fig4 ). the importance of the sequence β93 - 100 was recognised by previous workers who proposed that the variability in charge in this loop would act as a determinnant of specificity , with positive charge for lh / hcg as opposed to the negative charge found for fsh and tsh . the disulphide 93 - 100 which forms this loop has been shown to be important for activity . the structure reveals that the determinant loop is surface oriented and held in place by a short stretch of parallel β - sheet between α53 - 56 and β97 - 102 . β98 thr is buried at the interface with α , and its importance in subunit association has been implicated from mutagenesis studies . residues implicated in receptor binding from site - directed mutagenesis ( arg 94 , arg 95 , ber 96 and asp 99 ) are surface accessible in the dimer . thr 97 , which is generally insensitive to mutagenesis , is also surface accessible but does not contribute to the same surface as the other receptor binding residues . however , mutagenesis of this residue to asp destroys receptor binding indicating that a negative charge in this region is not tolerated . much attention has been focused on the longest inter - cysteine loop β35 - 58 which has been shown to stimulate steroidogenesis in rat leydig cells and inhibit binding of whole hcg half - maximally at concentrations of 10 - 5 m . the crystal structure reveals an unusual loop with the n - terminal residues involved in the heterodimer formation . the conserved residue val 44 is entirely buried at the α / β interface , and thr 40 , met 41 , thr 42 , leu 45 and gln 46 also make significant contributions to the buried surface at the diner interface . β35 - 45 adopts a β strand conformation and forms a short stretch of anti - parallel β - sheets with α27 - 31 . there is a poorly defined section between β46 and 50 . pro 50 forms a turn and the section β50 - 57 forms an almost linear structure . val 55 and 56 pass between cys 90 and cys 93 and are partially buried . the side chain of the conserved residue gln 54 is buried and forms hydrogen bonds to the mainchain of β41 and α87 . the hydrophobic residues 50 - 53 are all exposed to solvent , as are residues 47 - 50 . mutagenesis of arg 43 to leu ( as in fsh ) in the whole hcg , or replacement of it by ala or asp in synthetic ( 38 - 57 ) peptides either significantly diminishes binding or eliminates it in the case of the peptides . retention of partial activity is yet more evident for multiple binding sites over the hormone surface . from the orientation of arg 43 in the structure a possible role in receptor binding is to stabilise the conformation of pro 50 and the long loop region . the c - terminus of the α - subunit has been highly conserved throughout evolution , the five amino acid residues of the carboxy - terminus , tyr - tyr - his - lys - ser - cooh are identical in 13 of the 19 available amino acid sequences and his 90 and lys 91 are invariant . it has been shown that carboxypeptidase digestion of both hcgα and lhα , while not detrimental to subunit assembly , essentially eliminates receptor binding of the diner . des -( 89 - 92 ) hcgα has been shown to form an active heterodimer with the β - subunit but with reduction of both receptor binding and initiation of steroidogenesis . the c - terminus of the α subunit is located in close proximity to both the ` long loop ` and the ` determinant loop ` of the β subunit , which would imply a single major receptor binding site . although residues 88 and 89 are poorly defined and final three residues are not seen in the structure , it can be deduced that they extend out beside the ` determinant loop ` as shown in fig4 and it seems likely that the invariant his 90 and lys 91 are involved in receptor binding . appropriate glycosylation of gonadotropins has been shown to be essential for full expression of their biological activity . chemically deolycosylated hcg ( hf - hcg ) binds to receptors with slightly increased affinity but the potency in activating adenylate cyclase and steroidogenesis is either greatly reduced or abolished . the structure provides an explanation why the carbohydrate at α52 asn is critical to the biological activity of the hormone . the carbohydrate is positioned at the dimer interface and is about 4 å away from the determinant loop and the observation that removal of carbohydrate increases receptor binding can be explained by the close proximity of this carbohydrate chain to the potential receptor binding site of the hormone . it can be envisaged that its position , size and flexibility could be antagonistic to a completely peptide based receptor binding . site - directed mutagenesis studies have shown that removal of both β - subunit n - glycosylation sites does not cause drastic loss of activity , but this mutant is not able to produce the same maximal steroidogenic activity in mouse leydig tumor cells ( ma - 10 cells ). the two asn residues are very close to each other and located 20 å from α52 asn which would seem to indicate a different site of action , but modelling with the complete complex carbohydrate shows that in fact the carbohydrates would be quite close to each other and form a band of carbohydrate around the molecule . fig8 shows a computer visual display means in the form of a computer monitor 2 connected to a computer ( not shown ) and a keyboard 4 also connected to the computer . the computer is loaded with a commercially - available software package comprising a plurality of programs operating together to provide a three - dimensional simulated model of the hcg molecule and to simulate the affect any chemical changes made to the structure thereof may have on the three - dimensional shape thereof . on the monitor is displayed the three - dimensional shape of hcg according to fig5 c . fig9 is a block diagram of the computer used to store and execute the three - dimensional molecular simulation software , which comprises a memory which may typically include a random access memory and optionally a read only memory . a hard disc memory is also provided onto which the software is loaded and stored , and into which the input coordinates data and inputted changes in chemical structure may be stored . the three - dimensional representations of the original hormone and hormone analogue may also be stored on the disc . data processing operations are carried out in the processor which receives information from the memory and the disc , and also receives input information from the input means such as a keyboard . the processor also generates three - dimensional representions of the molecules and displays these on the visual display means e . g . computer monitor . a general protocol for applying the invention is schematically depicted in fig1 . by inspection of the 3d - structure of hcg it is possible to indicate optimal positions where the consensus sequence for n - glycosylation ( asn - x - ser / thr , x not pro ) can be introduced . the selection criteria have been described in the text above . based on the 3d - structure of hcg homology built models of fsh and lh may also be constructed ; thus allowing optimal positions in these gonadotropins to be determined . by one single point mutation extra glycosylation sites are created in 1 !- 8 ! as follows : in order to further reduce the size of the glycoprotein hormone family , non - essential hairpins are deleted . inspection of the 3d - structure and knowledge of the structure - activity relationships indicates that in particular the beta strand formed by residues 57 to 90 of the beta subunit is an attractive candidate for a truncation . apart from specifying which residues can be deleted also several additional residues are needed to join the separated regions and to form the new hairpin loop . for this purpose amino acids that are known to frequently occur in hairpins are used . for several types of beta turns , preference exists for the amino acid composition at the 3 and 4 positions in the loop . thus , taking all of this into account , the following structure is produced : 9 ! beta 1 - 59 !- asp - ser - asn - beta 86 - 108 ! combined with alpha . the modified hcg analogue produced by the computer software to incorporate these chemical changes and the three - dimensional structure displayed on the computer monitor is shown in fig1 . 1 . manjunath , p . & amp ; sairam , m . r . j . biol , chem . 257 , 7109 - 7115 ( 1982 ). 2 . harris , d . c ., machin , k . j ., evin , g . m ., morgan , f . j . & amp ; isaacs , n . w . j . biol . chem 264 , 6705 - 6706 ( 1989 ). 3 . lustbader , j . w ., birken , s ., pileggi , n . f ., kolks , m . a . g ., pollack , s ., cuff , m . e ., yang , w ., hendrickson , w . a . & amp ; canfield , r . e . biochemistry 28 , 9239 - 9234 ( 1989 ). 4 . tyan , r . j ., keutmann , h . t ., charlesworth , m . c ., mccormick , d . j ., milius , r . p ., calvo , f . o . & amp ; vutyavanich , t . rec . prog . in hormone res . 43 , 383 - 429 ( 1987 ). 5 . murray - rust , j ., mcdonald , m . q ., blundell , t . l ., hosang , m ., oefner , c ., winkler , f . & amp ; bradshaw , r . s ., structure 1 , 153 - 159 ( 1993 ). 7 . mcdonald , n . w ., lapatto , r ., murray - rust , j ., gunning , j ., wlodawer , a . & amp ; blundell , t . l . nature 354 , 411 - 414 ( 1991 ). 8 . schlunegger , m . p . & amp ; grutter , m . g . j . mol ., biol . 231 , ( 1993 ). 9 . oefner , c ., d &# 39 ; arcy , a ., winkler , f . k ., eggiman , b & amp ; hosang , m . embo j . 11 , 3932 - 3926 ( 1992 ). __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 92 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( iii ) hypothetical : no ( vi ) original source :( a ) organism : homo sapiens ( b ) strain : alpha - subunit human chorionic gonadotropin ( xi ) sequence description : seq id no : 1 : alaproaspvalglnaspcysproglucysthrleuglngluasnpro151015phepheserglnproglyalaproileleuglncysmetglycyscys202530pheserargalatyrprothrproleuargserlyslysthrmetleu354045valglnlysasnvalthrsergluserthrcyscysvalalalysser505560tyrasnargvalthrvalmetglyglyphelysvalgluasnhisthr65707580alacyshiscysserthrcystyrtyrhislysser8590 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 145 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( iii ) hypothetical : no ( iv ) anti - sense : no ( vi ) original source :( a ) organism : homo sapiens ( b ) strain : beta - subunit human chorionic gonadotropin ( xi ) sequence description : seq id no : 2 : serlysgluproleuargproargcysargproileasnalathrleu151015alavalglulysgluglycysprovalcysilethrvalasnthrthr202530ilecysalaglytyrcysprothrmetthrargvalleuglnglyval354045leuproalaleuproglnvalvalcysasntyrargaspvalargphe505560gluserileargleuproglycysproargglyvalasnprovalval65707580sertyralavalalaleusercysglncysalaleucysargargser859095thrthraspcysglyglyprolysasphisproleuthrcysaspasp100105110proargpheglnaspserserserserlysalaproproproserleu115120125proserproserargleuproglyproseraspthrproileleupro130135140gln145__________________________________________________________________________