we expressed a homolog of eukaryotic sterol reductases from the methanotrophic bacterium methylomicrobium alcaliphilum 20z ( masr1 , gi : 503913803 ) . its cdna was cloned into pet-21b ( novagen ) with an n - terminal 8-his tag . the transformed c43(de3 ) ( lucigen ) cells were grown to an optical density of 1.0 at a600 nm and induced with 0.2 mm isopropyl--d - thiogalactopyranoside ( iptg ) . cells were disrupted using a french press with 2 passes at 15,000 psi , in a buffer a containing 25 mm tris - cl , ph 8.0 , and 150 mm nacl . after a low speed centrifugation , the resulting supernatant was centrifuged at high speed to sediment a membrane fraction , which then was incubated in buffer a with 2% ( w / v ) n - dodecyl--d - maltopyranoside ( ddm , anatrace ) for 1 hour at 4 c . the lysate was centrifuged again and the supernatant was loaded onto ni - nta affinity column ( qiagen ) . after washing twice , the protein was eluted with 25 mm tris - cl , ph8.0 , 150 mm nacl , 300 mm imidazole , and 0.1% ddm , and concentrated by centricon for subsequent gel filtration ( superdex-200 , ge healthcare ) in buffer a with 0.4% ( w / v ) n - nonyl--d - glucopyranoside ( -ng , anatrace ) . selenomethionine ( se - met ) labeled protein was purified similarly with the exception that 1 mm tris [ 2-carboxyethyl ] phosphine ( tcep ) was included during the purification process . before crystallization , the crystals appeared after 5 days in the well buffer containing 0.1 m tris - cl ph 7.0 , 0.2 m nh4ac , 30% ( v / v ) pentaerythritol ethoxylate ( 15/4 eo / oh ) . ddm was added into crystallization buffer at 1% ( v / v ) final concentration to improve diffraction . selenomethionine ( se - met ) labeled protein was crystallized in the same buffer supplemented with 6 mm dtt . platinum derivatives were obtained by soaking native crystals for 12  hours in mother liquor plus 10  mg / ml k2pt(no2)4 . all crystals were directly flash - frozen in a cold nitrogen stream at 100 k. the data were collected at national synchrotron light source ( nsls ) beamline x29 . owing to the anisotropic diffraction properties , the outlier reflections were rejected based on extreme - value wilson statistics using the program xtriage in the phenix package . the anomalous signal in the semet - derivative data was further magnified with the local - scaling algorithm using the program solve . the identified sites were refined and the initial phases were generated in the program phaser with the sad experimental phasing module . two - fold ncs averaging along with solvent flattening and histogram matching was performed using dm . introduction of additional selenium anomalous scatterers by selective mutation , and preparation of platinum derivatives confirmed the correctness of the atomic model . the homology models of human lbr and human dhcr7 was generated by the program modeller on the basis of the structure of masr1 in which the n - terminal regions ( 1 - 200 of lbr and 1 - 58 of dhcr7 ) were excluded because of low sequence conservation ( extended data fig . wild - type and mutant masr1 and scerg24 were subcloned into the ura3 shuttle vector pcm190 ( euroscarf , germany ) . the plasmids were introduced in erg24 deficient saccharomyces cerevisiae strain y11164 ( euroscarf ) by electroporation . a single colony was picked from a ura selective plate . for the yeast rescue assay , the yeast was grown on ura plates either in the absence or the presence of sub - inhibitory concentrations of cycloheximide ( 20 ng / ml ) at 30c for 24 to 48 hours . the cdna encoding human delta-14 sterol reductase ( c14sr ) was subcloned into pcmv - sport6 ( open biosystems ) . wild type and mutant masr1 were subcloned into pegfp - n1 ( clontech ) without the egfp - tag . hek293 cells were grown in a 5% co2 incubator at 37c in dmem supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 100 u / ml penicillin , and 0.1 mg / ml streptomycin . cells were cultured in 10-cm petri dishes to 80% confluence and transfected with the plasmids , using lipofectamine 2000 ( life technologies ) . after 48 hours , transfected cells were recovered and the washed cell pellet was resuspended in pbs containing complete protease inhibitor cocktail ( sigma - aldrich ) . cells were lysed by sonication three times for 10 seconds on ice . after a low speed centrifugation , the isolated membrane fraction was resuspended in 10 mm kpo4/0.5 mm edta ( ph 7.4 ) and freezed in aliquots for further analyses . proteins of the membrane fraction were separated by sds - page , blotted on pvdf and probed with mouse monoclonal anti - flag m2 ( sigma - aldrich ) and peroxidase - conjugated goat anti - mouse ( santa cruz ) . delta-14 reductase activity was assayed in the membrane fractions obtained from transfected cells ( 0.25 mg protein per assay ) using 5-cholesta-8,14-dien-3-ol ( c27 ) as a substrate . after the addition of 5-cholestane ( 5 g ) as an internal standard , sterols were extracted with petroleum ether , desiccated under nitrogen stream and converted to trimethylsilyl derivatives using n , o - bis ( trimethylsilyl ) trifluoroacetamide ( bstfa ) and pyridine ( 1:1 , v / v ) . gas chromatography  mass spectrometry ( gc - ms ) analysis was performed in multiple ion detection mode using a varian gc - ms 2000 apparatus with a varian cp - sil8 cb low bleed capillary column . sterol retention times were : 15.31 min , 5-cholestane ( m / z 372 ) ; 19.90 min , cholesterol ( m / z 458 ) ; 20.16 min , 5-cholesta-8,14-dien-3-ol ( c27 , m / z 456 ) ; 20.34 min , 5-cholesta- 8-en-3-ol ( c27 , m / z 458 ) . we expressed a homolog of eukaryotic sterol reductases from the methanotrophic bacterium methylomicrobium alcaliphilum 20z ( masr1 , gi : 503913803 ) . its cdna was cloned into pet-21b ( novagen ) with an n - terminal 8-his tag . the transformed c43(de3 ) ( lucigen ) cells were grown to an optical density of 1.0 at a600 nm and induced with 0.2 mm isopropyl--d - thiogalactopyranoside ( iptg ) . cells were disrupted using a french press with 2 passes at 15,000 psi , in a buffer a containing 25 mm tris - cl , ph 8.0 , and 150 mm nacl . after a low speed centrifugation , the resulting supernatant was centrifuged at high speed to sediment a membrane fraction , which then was incubated in buffer a with 2% ( w / v ) n - dodecyl--d - maltopyranoside ( ddm , anatrace ) for 1 hour at 4 c . the lysate was centrifuged again and the supernatant was loaded onto ni - nta affinity column ( qiagen ) . after washing twice , the protein was eluted with 25 mm tris - cl , ph8.0 , 150 mm nacl , 300 mm imidazole , and 0.1% ddm , and concentrated by centricon for subsequent gel filtration ( superdex-200 , ge healthcare ) in buffer a with 0.4% ( w / v ) n - nonyl--d - glucopyranoside ( -ng , anatrace ) . selenomethionine ( se - met ) labeled protein was purified similarly with the exception that 1 mm tris [ 2-carboxyethyl ] phosphine ( tcep ) was included during the purification process . before crystallization , the protein solution was incubated with 2 mm nadph ( sigma - aldrich ) . the crystals appeared after 5 days in the well buffer containing 0.1 m tris - cl ph 7.0 , 0.2 m nh4ac , 30% ( v / v ) pentaerythritol ethoxylate ( 15/4 eo / oh ) . ddm was added into crystallization buffer at 1% ( v / v ) final concentration to improve diffraction . selenomethionine ( se - met ) labeled protein was crystallized in the same buffer supplemented with 6 mm dtt . platinum derivatives were obtained by soaking native crystals for 12  hours in mother liquor plus 10  mg / ml k2pt(no2)4 . owing to the anisotropic diffraction properties , the outlier reflections were rejected based on extreme - value wilson statistics using the program xtriage in the phenix package . the anomalous signal in the semet - derivative data the identified sites were refined and the initial phases were generated in the program phaser with the sad experimental phasing module . two - fold ncs averaging along with solvent flattening and histogram matching was performed using dm . introduction of additional selenium anomalous scatterers by selective mutation , and preparation of platinum derivatives confirmed the correctness of the atomic model . the homology models of human lbr and human dhcr7 was generated by the program modeller on the basis of the structure of masr1 in which the n - terminal regions ( 1 - 200 of lbr and 1 - 58 of dhcr7 ) were excluded because of low sequence conservation ( extended data fig . wild - type and mutant masr1 and scerg24 were subcloned into the ura3 shuttle vector pcm190 ( euroscarf , germany ) . the plasmids were introduced in erg24 deficient saccharomyces cerevisiae strain y11164 ( euroscarf ) by electroporation . a single colony was picked from a ura selective plate . for the yeast rescue assay , the yeast was grown on ura plates either in the absence or the presence of sub - inhibitory concentrations of cycloheximide ( 20 ng / ml ) at 30c for 24 to 48 hours . the cdna encoding human delta-14 sterol reductase ( c14sr ) was subcloned into pcmv - sport6 ( open biosystems ) . wild type and mutant masr1 were subcloned into pegfp - n1 ( clontech ) without the egfp - tag . hek293 cells were grown in a 5% co2 incubator at 37c in dmem supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 100 u / ml penicillin , and 0.1 mg / ml streptomycin . cells were cultured in 10-cm petri dishes to 80% confluence and transfected with the plasmids , using lipofectamine 2000 ( life technologies ) . after 48 hours , transfected cells were recovered and the washed cell pellet was resuspended in pbs containing complete protease inhibitor cocktail ( sigma - aldrich ) . cells were lysed by sonication three times for 10 seconds on ice . after a low speed centrifugation , the isolated membrane fraction was resuspended in 10 mm kpo4/0.5 mm edta ( ph 7.4 ) and freezed in aliquots for further analyses . proteins of the membrane fraction were separated by sds - page , blotted on pvdf and probed with mouse monoclonal anti - flag m2 ( sigma - aldrich ) and peroxidase - conjugated goat anti - mouse ( santa cruz ) . delta-14 reductase activity was assayed in the membrane fractions obtained from transfected cells ( 0.25 mg protein per assay ) using 5-cholesta-8,14-dien-3-ol ( c27 ) as a substrate . after the addition of 5-cholestane ( 5 g ) as an internal standard , sterols were extracted with petroleum ether , desiccated under nitrogen stream and converted to trimethylsilyl derivatives using n , o - bis ( trimethylsilyl ) trifluoroacetamide ( bstfa ) and pyridine ( 1:1 , v / v ) . gas chromatography  mass spectrometry ( gc - ms ) analysis was performed in multiple ion detection mode using a varian gc - ms 2000 apparatus with a varian cp - sil8 cb low bleed capillary column . sterol retention times were : 15.31 min , 5-cholestane ( m / z 372 ) ; 19.90 min , cholesterol ( m / z 458 ) ; 20.16 min , 5-cholesta-8,14-dien-3-ol ( c27 , m / z 456 ) ; 20.34 min , 5-cholesta- 8-en-3-ol ( c27 , m / z 458 ) . conversion of lanosterol to cholesterol ( bloch pathway ) involves many reactions , some of which are catalyzed by c14sr , lbr and dhcr7 ( masr1 homologues , in red ) . c14sr , lbr and dhcr7 are homologues of nadph - dependent reductases that catalyze the reduction of the sterol double bonds indicated in the green circles . sequence alignment of masr1 with human c14sr , dhcr7 and c - terminal domain of lbr . invariant amino acids are highlighted in blue ( invariant in 3 of 4 proteins ) and purple ( invariant in all proteins ) . putative cholesterol hydroxyl group binding sites are highlighted in red , nadph binding sites are highlighted in cyan . masr1 can rescue the growth of a saccharomyces cerevisiae delta-14 sterol reductase erg24 ( yeast masr1 homolog ) deletion strain ( erg24 ) . erg24 yeast expressing wild - type masr1 , scerg24 and mutated masr1 from a ura3 shuttle vector can grow under ura selection ( upper panel ) . growth of yeast expressing masr1 , scerg24 and various mutated masr1 versions under in the presence of sub - inhibitory concentrations of cycloheximide ( 20ng / ml ) for 24 to 48 hours ( lower panel ) . the yeast expressing masr1 or scerg24 is able to grow in the presence of cycloheximide . r395a ( lane 8) corresponds to r583q in lbr which has been reported to lead to loss of activity in yeast . masr1 crystal and x - ray diffraction image . a , photograph of masr1 crystal . b , a representative x - ray diffraction image of masr1 crystals with various resolution rings indicated by the circles . anomalous difference fourier electron density . a , overview of the anomalous difference fourier map for selenium atoms in an asymmetric unit . left panel shows wild type se - met anomalous difference signals , right panel shows mutated se - met anomalous difference signals at 3 ( purple mesh ) . left panel shows wild type se - met anomalous difference signals , right panel shows mutated se - met anomalous difference signals at 3 ( blue mesh ) . d , a view of the anomalous difference fourier map for platinum atoms in an asymmetric unit . there are 4 platinum atoms binding to histidine residues in molecule a ( yellow ) , but there are 8 platinum atoms binding to 6 histidine and 2 methionine residues in molecule b ( red ) . e , an overall view of the 2fo - fc electron density , contoured at 2 , in one asymmetric unit . nadph binding pocket and interaction between trp274 and tyr387 of masr1 . a , the structure of nadph with the missing moiety in the masr1 structure indicated in the black circles . sa - omit map ( fo - fc densities , magenta mesh ) for nadph contoured at  level 2.0 . the right panel is an enlargement of the left panel ( same orientation as fig . 2a ) , rotated by 180. c , the rebuilded missing moiety ( purple ) of nadph in masr1 . d , the surface representation shows trp274 ( orange ) and tyr387 ( blue ) just located in the back of the sterol binding pocket . mfo - dfc map for an unidentified ligand ( blue mesh ) contoured at 2. comparison of masr1 structure with icmt structure . a , a comparison of masr1 ( gray and yellow ) and icmt ( cyan ) structure with sah bound ( pdb 4a2n ) . dali search shows the closest entry ( z - score of 7.5 ) to masr1 is the structure of isoprenylcysteine carboxyl methyltransferase ( icmt ) consisting of 5 transmembrane helices , which had 193 c atoms aligned to masr1 ( tms 6 - 10 and 2 ) with rmsd of 2.8 . both proteins have a similar cofactor - binding pocket ( magenta circle ) , although the sequence conservation is low . b , comparison of nadph and sah binding pockets of masr1 ( gray ) and icmt ( cyan ) . the orientation of adenine - ribose moiety of sah and napdh is similar with respect to the coordinating tyrosine residues in the cofactor pockets of these two enzymes . data collection statistics for the masr1 mutants i151 m , l304 m , and pt - derivatives .
sterols are essential biological molecules in the majority of life forms . 
 sterol reductases1 including delta-14 sterol reductase ( c14sr ) , 7-dehydrocholesterol reductase ( dhcr7 ) and 24-dehydrocholesterol reductase ( dhcr24 ) reduce specific carbon - carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis . 
 lamin b receptor2 ( lbr ) , an integral inner nuclear membrane protein , also contains a functional c14sr domain . here 
 we report the crystal structure of a delta-14 sterol reductase ( masr1 ) from the methanotrophic bacterium methylomicrobium alcaliphilum 20z , a homolog of human c14sr , lbr , and dhcr7 , with the cofactor nadph . 
 the enzyme contains 10 transmembrane segments ( tm ) . 
 its catalytic domain comprises the c - terminal half ( containing tm6 - 10 ) and envelops two interconnected pockets , one of which faces the cytoplasm and houses nadph , while the other one is accessible from the lipid bilayer . 
 comparison with a soluble steroid 5-reductase structure3 suggests that the reducing end of nadph meets the sterol substrate at the juncture of the two pockets . 
 a sterol reductase activity assay proves masr1 can reduce the double bond of a cholesterol biosynthetic intermediate demonstrating functional conservation to human c14sr . 
 therefore , our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in dhcr7 and lbr for inborn human diseases .