complex i was purified from b. taurus heart mitochondrial membranes by solubilisation and anion exchange chromatography in n - dodecyl--d - maltoside ( ddm ) , and size - exclusion chromatography in ddm or 7-cyclohexyl-1-heptyl--d - maltoside ( cymal 7 ) as described previously . aliquots of complex i ( 3 l , 3 - 4.5 mg ml ) were applied to glow - discharged holey - carbon quantifoil r 0.6/1 grids , blotted for 15 - 18 s , then plunge - frozen in liquid ethane using an environmental plunge - freeze apparatus . the grids were transferred into cartridges , loaded into an fei titan krios electron microscope , and images were recorded at 2 - 5 m underfocus on a falcon ii cmos ( complementary metal oxide semiconductor ) direct electron detector at 300 kev at 81,495 magnification ( nominally 47,000 ) , with the specimen temperature at 186 c using the epu software ( extended data fig . the detector pixel size of 14 m corresponds to a sampling density of ~1.72  pixel . each image was exposed for 4 s ( total dose ~64 e  ) and 72 frames were captured . for tilt - pair analysis , the same area was imaged twice , at 0 for 0.8 s and then at 10 for 2.0 s , and analysed with frealign . an initial data set for complex i in ddm was obtained by manually picking particles with ximdisp . 7630 particles , from 366 micrographs , were used to generate initial maps in eman2 . particles were boxed in 280  280 pixels and contrast transfer function ( ctf ) parameters estimated internally . reference - free classification was performed using the default eman2 parameters , and classes with distinct orientations selected ( see extended data fig . the initial model that best matched the class - averages was selected and two cycles of refinement performed in eman2 . subsequently , comparison of the model with the structure of complex i from t. thermophilus ( 4hea.pdb ) suggested that it had the wrong hand ; this observation was verified using tilt - pair analysis and corrected ( extended data fig . all further refinements were performed in relion , starting with maps that were low - pass filtered to 60  . typical micrographs prepared from complex i in cymal 7 exhibited , on average , twice as many particles ( ~40 per micrograph ) than those from complex i in ddm , so a larger data set was collected using cymal 7 . the reason for the difference in particle distribution is not clear  it may simply be a product of the grid preparation and freezing protocols . the class averages were used as a reference to pick particles automatically using relion but many false positives were included , so all the images were inspected manually and particles too close to each other , aggregates , and ice contaminants , were deleted . the final data set contained 45,618 particles from 1,154 micrographs . the ctf was determined with ctffind3 using the images summed from all 72 frames . subsequently , refinement was performed using frames 1 - 32 of each image ( the last 40 frames were discarded ) , to produce a map with resolution of 5.86  and orientational accuracy of 1.2. to check for overfitting , phases were randomized beyond 10  on individual images ( frames 1 - 32 ) , followed by refinement as for the normal images . the results clearly show the presence of information beyond 10  ( extended data fig . 2 ) . note that relion divides the data set into two halves at the initial step and calculates the resolution using a gold - standard fourier shell correlation ( fsc ) , so the phase randomisation procedure serves here only as an additional control . modelling of the beam - induced movement of the complex i particles ( using a running average of 11 movie frames in relion ) provided a modest improvement in resolution to 5.16  . the parameters from this analysis were then used to carry out a per - frame reconstruction in relion ( particle - polish ) , and a b - factor weighting was applied to each frame , resulting in a 4.8  map . the b - factor weighted particles were subjected to 25 iterations of 3d classification into 4 classes ; this separated a major class containing 55% of the particles from smaller classes of 24 , 14 and 6% . difference maps revealed minor localised variations in some of the peripheral regions of the molecule , but no large - scale conformational variation was observed . the major class with 25,492 particles was refined and , after post - processing with relion , a shape - mask , correction for the modulation transfer function ( mtf ) of the detector and a b - factor of 152 were applied , and filtered to 4.95  resolution ( extended data fig 2c , note that the magnification and ctf values have not been refined ) . despite containing a lower number of particles , the maps from this major class and the whole data set were comparable . analysis of the local resolution by resmap showed that the core sections of the molecule , particularly the tmhs , have higher resolutions than the peripheral sections , and that ( as expected ) the detergent - phospholipid belt is at lower resolution ( extended data fig . all the models described have been built as polyalanine chains , except for the residues that coordinate the fes clusters . note that the present model has not been refined , so it inevitably contains some errors and inaccuracies . examples of the model fitted to the electron density are shown in extended data fig . 3 , and figures were created using the pymol molecular graphics system ( schrdinger , llc ) or ucsf chimera ( http://www.cgl.ucsf.edu/chimera/ ) . the seven core hydrophilic subunits of b. taurus complex i were modelled initially using the coordinates of t. thermophilus complex i ( 4hea.pdb ) as a template , trimmed where the densities were ambiguous , and adjusted manually . fes clusters were located using the highest peak densities in the unsharpened map , and the subunits were built around them . the 24 and 51 kda subunits were easily built as they are well conserved and the connectivity in the densities is clearly resolved . the 49 kda subunit has dominant secondary structures and , except for the n - terminal peptide , could be completely traced . similarly , the psst and tyky subunits , with three fes clusters , were readily built . the central part of the 30 kda subunit , containing a mixture of -helices and -strands , could be traced , but the path of the long unstructured c - terminus is unclear . the small domain containing the three fes clusters is well resolved and could be traced easily using the t. thermophilus model , but significant portions of the large , peripheral domain have poor density , low secondary structure content and low sequence similarity to t. thermophilus , and so could not be traced confidently . the seven core subunits in the membrane domain could all be readily traced , except for a few loop regions , and assigned using their similarity to the t. thermophilus subunits . the long transverse helix at the c - terminus of subunit nd5 is well ordered and extends over nd4 and nd2 . in better resolved regions of the map , protruding densities that are likely to be side chains of aromatic residues are observed ( see extended data fig . 3 ) , and these features , along with secondary structure information and sequence alignments , were used to produce an optimised assignment for the b. taurus residue numbers in the modelled subunits ( extended data table 2 ) , for use as a guide to the positions of individual residues . once the electron density for the core subunits had been assigned , models for the tmhs of the supernumerary subunits were built . a total of 18 tmhs were modelled , and when the density was clear they were extended . connectivity was observed between four tmhs adjacent to subunit nd4 so they were combined into a single chain . to aid in supernumerary subunit assignments , the secondary structure of each subunit was predicted using psipred and tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) ( extended data table 4 ) . known structures of soluble proteins with high homology to the complex i supernumerary subunits were identified by hhpred and used to build homology models in modeller and swissmodel ( extended data table 5 ) . regions of the density map with features corresponding to the predicted structures were located manually . long loop regions were trimmed , then the models were placed in the density , jiggle fit in coot was used to find the best fit , and the models were adjusted manually . most of them are located close to tmhs from the supernumerary subunits , but the connectivity to them is not clear ; a higher resolution map will be necessary to assign these helices to their respective subunits . a ) typical micrograph of complex i particles imaged after freezing in vitreous ice on a holey - carbon grid . the size of each box is 280 pixels and the 2d classification was made in relion . 100 complex i particles from eight image pairs , recorded with a relative tilt angle of 10 , were extracted and subjected to tilt - pair analysis with frealign . the outer radius of the plot is 40 and the orange circle centered at the expected tilt angle has a radius of 6. phases that are beyond 10  in each of the micrographs used in the final data set ( frames 1 - 32 ) were randomised , and then refinement was performed as for a normal data set ( fsc summed image corresponding to frames 1 - 32 ) . as expected , the graph shows a drop in the fourier shell correlation ( fsc ) curve at 10  , validating the presence of information beyond 10  in the images . note that the use of gold - standard refinement procedures in relion prevents any overfitting , and this test was done only as an additional control . d ) fsc curves of the final map and of the model versus the map . the curve in red is the gold - standard fsc of the final map ( after classification ) and the resolution at fsc = 0.143 is ~4.95  . the curve in cyan is the fsc between the final map and the model , and at fsc = 0.5 the resolution is 6.7  . note that the present model is not complete since it is only a polyalanine model without any side chains , and loop regions in a number of subunits have not been modelled . the left - hand panel ( with lower density threshold ) shows that the detergent / phospholipid belt is of lower resolution , and the protein regions of the map show resolution distributed from 5 to 6  . in the right - hand panel the map is shown at higher density threshold , so the detergent / phospholipid belt is not visualised . some of the interior parts of the map have resolution of 4.8 - 5  . a ) nd2 is shown from the membrane plane , high - lighting the densities for three aromatic sidechains and one of the helix - breaking loops . c ) the density for a [ 4fe-4s ] cluster and surrounding protein is shown in the psst subunit . d ) a region of the 49 kda subunit shows a well resolved -helical stretch and aromatic side - chains , and the -strands are beginning to be resolved . e ) subunit b8 is an example of a supernumerary subunit in a peripheral region of the molecule . f ) in the 39 kda subunit density consistent with a bound nucleotide is observed , in a similar position to in homologous structures , and as expected from analysis of y. lipolytica complex i. however , the present resolution of the map precludes the inclusion of this nucleotide in the final model . the core subunits from b. taurus are in blue , and from t. thermophilus ( 4hea.pdb ) in orange . top : the nd2 , nd4 and nd5 domain is rotated in b. taurus relative to in t. thermophilus , increasing the curvature in the b. taurus membrane domain . the complex is viewed along the 11 rotation vector ( orange ) that maps the t. thermophilus nd2 , nd4 and nd5 domain to the b. taurus domain , along with a small 5  translation to superimpose the domain centres . correspondingly , the nd3 , nd4l and nd6 domains are superimposed by a 4 rotation and a 1  translation . rotation of nd2 , 4 and 5 about the long axis of the domain , as noted for y. lipolytica , is not observed . bottom : the nadh dehydrogenase domain containing the 51 and 24 kda subunits is rotated by 23 and translated by 14  in b. taurus , relative to in t. thermophilus , causing the fes chains to diverge as the distance from nd1 increases . correspondingly , the 49 kda , psst and tyky subunits are superimposed by a 6 rotation and a 2  translation . the structures were analysed using superpose from the ccp4 suite and the 75 kda and 30 kda subunits were not included due to their lower structural conservation . a ) the structure of each subunit from t. thermophilus ( wheat , 4hea.pdb ) has been superimposed separately on its corresponding subunit from b. taurus ( coloured as labelled ) with the transverse helix plus tmh16 of nd5 also aligned separately . the complexes are viewed from behind nd1 ( top ) , from the side ( middle ) and from the matrix ( bottom , nd subunits only ) . b ) observed differences in the structures of the core subunits of b. taurus and t. thermophilus complexes i. grey , conserved structure from b. taurus and t. thermophilus ( 4hea.pdb ) ; red , structural elements present only in t. thermophilus ; blue , structural elements present only in b. taurus . the c - terminal domain of the 75 kda subunit is not resolved in b. taurus , but its structure is clearly different to in t. thermophilus . table 1reference table for the nomenclature of the core subunits of complex iin the text the names of the subunits from b. taurus are used , with the names from the human enzyme presented alongside as appropriate.domainchain identifier 
 bos taurus 

 homo sapiens 

 yarrowia lipolytica 

 thermus thermophilics 

 escherichia coli 
 hydrophilic domaing75 kdandufs1nuamnqo3nuogf51 kdandufv1nubmnqo1nuofd49 kdandufs2nucmnqo4nuocdc30 kdandufs3nugmnqo5e24 kdandufv2nuhmnqo2nuoebpsstndufs7nukmnqo6nuobitykyndufs8nuimnqo9nuol 

 membrane domainhnd1nd1nu1mnqo8nuohnnd2nd2nu2mnqo14nuonand3nd3nu3mnqo7nuoamnd4nd4nu4mnqo13nuomlnd5nd5nu5mnqo12nuoljnd6nd6nu6mnqo10nuojknd4lnd4lnulmnqo11nuok in the text the names of the subunits from b. taurus are used , with the names from the human enzyme presented alongside as appropriate . table 2summary of the models of the core subunits of b. taurus complex isubunittotal residues*modelled residuespoorly resolved / uncertain residue numberingunresolved residuesunresolved elements ( > 10 residues)%modelled%identicalrmsdnd13183 - 200219 - 242253 - 3151 - 2201 - 218243 - 252316 - 318matrix loop ( tmh 5 - 6)ims loop ( tmh 6 - 7)90%(285/318)42%(132/318)1.60 nd23472 - 300320 - 346tmh111301 - 319347matrix loop ( tmh 10 - 11)94%(326/347)25%(86/347)2.08 nd31152 - 2352 - 112124 - 51113 - 115matrix loop ( tmh 1 - 2)72%(83/115)27%(31/115)2.05 nd44593 - 415430 - 455tmh141 - 2416 - 429456 - 459matrix loop ( tmh 13 - 14)96%(439/459)24%(111/459)2.20 nd4l981 - 8485 - 98matrix loop ( c - terminus)86%(84/98)21%(21/98)2.66 nd56064 - 2228 - 358363 - 400408 - 466487 - 513520 - 604tmh1tmh13 & tmh14tmh15transverse helix & tmh161 - 323 - 27359 - 362401 - 407467 - 486514 - 519605 - 606matrix loop ( tmh 1 - 2)matrix loop ( tmh 11 - 12)ims loop ( tmh 12 - 13)ims loop ( tmh 14 - 15)tmh 15 to transverse helix92%(558/606)31%(187/606)2.53 nd61752 - 7685 - 107140 - 172tmh5177 - 84108 - 139173 - 175matrix loop ( tmh 3 - 4)ims loop ( tmh 4 - 5)75% ( 131/175)16% ( 28/175)1.83  

 75 kdandufs17048 - 125136 - 318326 - 347367 - 400404 - 410425 - 495525 - 530542 - 627the large domain ( 222 - 704 ) is generally poorly resolved.the sequence alignment isweak and the secondary structurecontent low . residues 404 - 629are particularly poorly resolved.1 - 7126 - 135319 - 325348 - 366400 - 403411 -424496 - 524531 -541628 - 704probable loop regionprobable loop regionprobable loop regionprobable loop regionprobable subdomain75% ( 527/704)1 - 221 : 92%222 - 704 : 67%27% ( 189/704)1 - 221 40%222 - 704 : 21%1.96 1 - 221 : 1.57 222 - 704 : 2.11 51 kdandufv144431 - 441flavin and nadh binding site ( 63 - 72 , 99 - 104 , 181 - 189 , 300 - 304 , 327 - 333)1 - 30442 - 444n - terminal peptide93% ( 411/444)43% ( 191/444)1.61 49 kdandufs243047 - 4303-strand -sheet ( 47 - 79)1 - 46n - terminal region89% ( 384/430)42% ( 179/430)1.41 30 kdandufs322815 - 168numbering uncertain to 72loop / -strand ( 73 - 83)1 - 14169 - 228n - terminal peptidec - terminal region68% ( 154/228)24% ( 54/228)1.66 24 kdandufv221720 - 178loop 126 - 1321 - 19179 - 217n - terminal peptidec - terminal region73% ( 159/217)27% ( 59/217)1.57 psstndufs717927 - 169loop 68 - 791 - 26170 - 179n - terminal peptide80% ( 143/179)49% ( 88/179)1.44 tykyndufs817615 - 1761 - 14n - terminal peptide92% ( 162/176)36% ( 63/176)1.89 *for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein,.the % identity and the rmsd ( root mean square deviation calculated using pdbefold ) are between the sequences and structures of the subunits of b. taurus and t. thermophilus ( 4hea.pdb ) complexes i. for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein , . the % identity and the rmsd ( root mean square deviation calculated using pdbefold ) are between the sequences and structures of the subunits of b. taurus and t. thermophilus ( 4hea.pdb ) complexes i. table 3distances between the redox cofactors in structural models of complex i 
 t. thermophilus 

 b. taurus 


 hydrophilic domain ( 2fug.pdb)complex i ( 4hea.pdb)complex i ( this work ) 

 cofactors*centreedgecentreedgecentreedgen1a - flavin15.412.315.913.115.913.1flavin - cluster 1 ( n3)12.57.612.27.312.27.2n1a - cluster 1 ( n3)22.119.422.319.721.118.0cluster 1 ( n3 ) - cluster 214.011.013.710.714.011.0cluster 1 ( n3 ) - cluster 317.413.817.113.418.414.5cluster 2 - cluster 313.510.713.09.912.79.7cluster 3 - cluster 412.28.512.48.612.88.7cluster 4 - cluster 516.814.016.513.616.814.0cluster 5 - cluster 612.19.412.19.312.19.3cluster 6 - cluster 7 ( n2)13.710.513.510.213.610.5cluster 1 ( n3 ) - cluster 7 ( n2)61.157.660.557.061.558.1*the [ 2fe-2s ] cluster in the 24 kda subunit ( known as n1a ) is on the other side of the flavin from the main cofactor chain . the [ 4fe-4s ] cluster in the 51 kda subunit ( known as n3 ) is the first cluster in the chain and the [ 4fe-4s ] cluster in subunit psst ( known as n2 ) is the last ( seventh ) cluster in the chain.the distances are in ngstroms , between the geometric centres of the fe and s cluster cores or the flavin isoalloxazine ring system ( centre ) , or between the centres of the two closest atoms ( edge ) as commonly used in calculations of electron transfer rates . distances are estimated to be accurate to within 1 .the position of the flavin in b. taurus is poorly resolved and has been approximated using its position in 4hea.pdb . the [ 2fe-2s ] cluster in the 24 kda subunit ( known as n1a ) is on the other side of the flavin from the main cofactor chain . the [ 4fe-4s ] cluster in the 51 kda subunit ( known as n3 ) is the first cluster in the chain and the [ 4fe-4s ] cluster in subunit psst ( known as n2 ) is the last ( seventh ) cluster in the chain . the distances are in ngstroms , between the geometric centres of the fe and s cluster cores or the flavin isoalloxazine ring system ( centre ) , or between the centres of the two closest atoms ( edge ) as commonly used in calculations of electron transfer rates . the position of the flavin in b. taurus is poorly resolved and has been approximated using its position in 4hea.pdb . sapiens subunit*subcomplexsequence informationpredicted tmhs10 kdandufv3i and i018 kdandufs4i and i015 kdandufs5i onlycx9c motif , intermembrane space013 kdandufs6i and ipfam zinc - finger motif cx8hx15cx2c0mwfendufa1i only1b8ndufa2i and i0b9ndufa3i only1b13ndufa5i and i0b14ndufa6i onlylyr motif0b14.5andufa7i and i0pgivndufa8i onlytwo cx9c motifs , pfam chch domain intermembrane space039 kdandufa9i onlyshort - chain dehydrogenase reductase family , nadp binding,042 kdandufa10i only ( low level)similarity to deoxynucleoside kinases0b14.7ndufa11i ( i at low level)3 or 4b17.2ndufa12i and i0b16.6ndufa13i and i1sdapndufab1both i iacyl - carrier protein,0mnllndufb1i0 ( or 1)agggndufb2i1 ( or 0)b12ndufb3i1b15ndufb4both i and i1sgdhndufb5i1b17ndufb6i1b18ndufb7icx9c motif , intermembrane space0ashindufb8i1b22ndufb9ilyr motif0pdswndufb10i0esssndufb11i1kfyindufc1none1b14.5bndufc2i ( low level)1 or 2*the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i.subcomplex i , which contains the seven hydrophilic core subunits and eight to nine supernumerary subunits , is considered to represent a significant portion of the hydrophilic domain of complex i. subcomplex i , which contains all the subunits of subcomplex i plus core subunit nd6 and nine to ten additional supernumerary subunits , represents the hydrophilic domain of complex i plus associated membrane subunits . subcomplex i , which contains nd4 and nd5 and 12 to 13 supernumerary subunits , represents part of the membrane domain.tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) and are presented as consensus values with less represented values in brackets and single outliers discarded . the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i. subcomplex i , which contains the seven hydrophilic core subunits and eight to nine supernumerary subunits , is considered to represent a significant portion of the hydrophilic domain of complex i. subcomplex i , which contains all the subunits of subcomplex i plus core subunit nd6 and nine to ten additional supernumerary subunits , represents the hydrophilic domain of complex i plus associated membrane subunits . subcomplex i , which contains nd4 and nd5 and 12 to 13 supernumerary subunits , represents part of the membrane domain . tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) and are presented as consensus values with less represented values in brackets and single outliers discarded . table 5summary of the models of the supernumerary subunits of b. tauruscomplex isubunitchain identifiertotal residues*pdb modelaligned residues%identicalmodelled residues%modelledrmsd42 kdandufa10o3202ocp21 - 25221% ( 49/232)22 - 5479 - 167172 - 210222 - 24157% ( 181/320)1.91 39 kdandufa9p3452q1w19 -32513% ( 41/307)19 - 185203 - 250285 - 32173% ( 252/345)2.52 18 kdandufs4q1332jya33 - 13337% ( 37/101)33 - 5976 - 11652% ( 69/133)2.42 13 kdandufs6r962jrr44 - 9634% ( 18/53)47 - 9349% ( 47/96)1.97 b8ndufa2s991s3a1 - 9994% ( 93/99)17 - 9681% ( 80/99)2.18 sdap-ndufab1t881f808 - 8436% ( 28/77)9 - 2328 - 8281% ( 71/88)1.18 sdap-ndufab1u881f808 - 8436% ( 28/77)8 - 8285% ( 75/88)1.36 b13ndufa5v1161 - 7161% ( 71/116)b14ndufa6w1281 - 7256% ( 72/128)pgivndufa8x1722lql35 - 11423% ( 18/80)1 - 8046% ( 79/172)2.40 b14.7ndufa11y1411 - 10675% ( 106/141)b16.6ndufa13z14433 - 9745% ( 65/144)b9ndufa3 or mwfendufa1a1541 - 2946% ( 71/154)b1 - 42*for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein 3.known structures with high homology to the complex i subunits were identified by hhpred.rmsd : root mean square deviation calculated using pdbefold between the structures of the subunits of b. taurus complex i and the pdb model structures.subunit with less certain assignment.residue numbers are arbitrary and not assigned to the sequence . for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein 3 . rmsd : root mean square deviation calculated using pdbefold between the structures of the subunits of b. taurus complex i and the pdb model structures . complex i was purified from b. taurus heart mitochondrial membranes by solubilisation and anion exchange chromatography in n - dodecyl--d - maltoside ( ddm ) , and size - exclusion chromatography in ddm or 7-cyclohexyl-1-heptyl--d - maltoside ( cymal 7 ) as described previously . aliquots of complex i ( 3 l , 3 - 4.5 mg ml ) were applied to glow - discharged holey - carbon quantifoil r 0.6/1 grids , blotted for 15 - 18 s , then plunge - frozen in liquid ethane using an environmental plunge - freeze apparatus . the grids were transferred into cartridges , loaded into an fei titan krios electron microscope , and images were recorded at 2 - 5 m underfocus on a falcon ii cmos ( complementary metal oxide semiconductor ) direct electron detector at 300 kev at 81,495 magnification ( nominally 47,000 ) , with the specimen temperature at 186 c using the epu software ( extended data fig . the detector pixel size of 14 m corresponds to a sampling density of ~1.72  pixel . each image was exposed for 4 s ( total dose ~64 e  ) and 72 frames were captured . for tilt - pair analysis , the same area was imaged twice , at 0 for 0.8 s and then at 10 for 2.0 s , and analysed with frealign . an initial data set for complex i in ddm was obtained by manually picking particles with ximdisp . 7630 particles , from 366 micrographs , were used to generate initial maps in eman2 . particles were boxed in 280  280 pixels and contrast transfer function ( ctf ) parameters estimated internally . reference - free classification was performed using the default eman2 parameters , and classes with distinct orientations selected ( see extended data fig . the initial model that best matched the class - averages was selected and two cycles of refinement performed in eman2 . subsequently , comparison of the model with the structure of complex i from t. thermophilus ( 4hea.pdb ) suggested that it had the wrong hand ; this observation was verified using tilt - pair analysis and corrected ( extended data fig . all further refinements were performed in relion , starting with maps that were low - pass filtered to 60  . typical micrographs prepared from complex i in cymal 7 exhibited , on average , twice as many particles ( ~40 per micrograph ) than those from complex i in ddm , so a larger data set was collected using cymal 7 . the reason for the difference in particle distribution is not clear  it may simply be a product of the grid preparation and freezing protocols . the class averages were used as a reference to pick particles automatically using relion but many false positives were included , so all the images were inspected manually and particles too close to each other , aggregates , and ice contaminants , were deleted . the final data set contained 45,618 particles from 1,154 micrographs . the ctf was determined with ctffind3 using the images summed from all 72 frames . subsequently , refinement was performed using frames 1 - 32 of each image ( the last 40 frames were discarded ) , to produce a map with resolution of 5.86  and orientational accuracy of 1.2. to check for overfitting , phases were randomized beyond 10  on individual images ( frames 1 - 32 ) , followed by refinement as for the normal images . the results clearly show the presence of information beyond 10  ( extended data fig . 2 ) . note that relion divides the data set into two halves at the initial step and calculates the resolution using a gold - standard fourier shell correlation ( fsc ) , so the phase randomisation procedure serves here only as an additional control . modelling of the beam - induced movement of the complex i particles ( using a running average of 11 movie frames in relion ) provided a modest improvement in resolution to 5.16  . the parameters from this analysis were then used to carry out a per - frame reconstruction in relion ( particle - polish ) , and a b - factor weighting was applied to each frame , resulting in a 4.8  map . the b - factor weighted particles were subjected to 25 iterations of 3d classification into 4 classes ; this separated a major class containing 55% of the particles from smaller classes of 24 , 14 and 6% . difference maps revealed minor localised variations in some of the peripheral regions of the molecule , but no large - scale conformational variation was observed . the major class with 25,492 particles was refined and , after post - processing with relion , a shape - mask , correction for the modulation transfer function ( mtf ) of the detector and a b - factor of 152 were applied , and filtered to 4.95  resolution ( extended data fig 2c , note that the magnification and ctf values have not been refined ) . despite containing a lower number of particles , the maps from this major class and the whole data set were comparable . analysis of the local resolution by resmap showed that the core sections of the molecule , particularly the tmhs , have higher resolutions than the peripheral sections , and that ( as expected ) the detergent - phospholipid belt is at lower resolution ( extended data fig . all the models described have been built as polyalanine chains , except for the residues that coordinate the fes clusters . note that the present model has not been refined , so it inevitably contains some errors and inaccuracies . examples of the model fitted to the electron density are shown in extended data fig . 3 , and figures were created using the pymol molecular graphics system ( schrdinger , llc ) or ucsf chimera ( http://www.cgl.ucsf.edu/chimera/ ) . the seven core hydrophilic subunits of b. taurus complex i were modelled initially using the coordinates of t. thermophilus complex i ( 4hea.pdb ) as a template , trimmed where the densities were ambiguous , and adjusted manually . fes clusters were located using the highest peak densities in the unsharpened map , and the subunits were built around them . the 24 and 51 kda subunits were easily built as they are well conserved and the connectivity in the densities is clearly resolved . the 49 kda subunit has dominant secondary structures and , except for the n - terminal peptide , could be completely traced . similarly , the psst and tyky subunits , with three fes clusters , were readily built . the central part of the 30 kda subunit , containing a mixture of -helices and -strands , could be traced , but the path of the long unstructured c - terminus is unclear . the small domain containing the three fes clusters is well resolved and could be traced easily using the t. thermophilus model , but significant portions of the large , peripheral domain have poor density , low secondary structure content and low sequence similarity to t. thermophilus , and so could not be traced confidently . the seven core subunits in the membrane domain could all be readily traced , except for a few loop regions , and assigned using their similarity to the t. thermophilus subunits . the long transverse helix at the c - terminus of subunit nd5 is well ordered and extends over nd4 and nd2 . in better resolved regions of the map , protruding densities that are likely to be side chains of aromatic residues 3 ) , and these features , along with secondary structure information and sequence alignments , were used to produce an optimised assignment for the b. taurus residue numbers in the modelled subunits ( extended data table 2 ) , for use as a guide to the positions of individual residues . once the electron density for the core subunits had been assigned , models for the tmhs of the supernumerary subunits were built . a total of 18 tmhs were modelled , and when the density was clear they were extended . connectivity was observed between four tmhs adjacent to subunit nd4 so they were combined into a single chain . to aid in supernumerary subunit assignments , the secondary structure of each subunit was predicted using psipred and tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) ( extended data table 4 ) . known structures of soluble proteins with high homology to the complex i supernumerary subunits were identified by hhpred and used to build homology models in modeller and swissmodel ( extended data table 5 ) . regions of the density map with features corresponding to the predicted structures were located manually . long loop regions were trimmed , then the models were placed in the density , jiggle fit in coot was used to find the best fit , and the models were adjusted manually . most of them are located close to tmhs from the supernumerary subunits , but the connectivity to them is not clear ; a higher resolution map will be necessary to assign these helices to their respective subunits . a ) typical micrograph of complex i particles imaged after freezing in vitreous ice on a holey - carbon grid . the size of each box is 280 pixels and the 2d classification was made in relion . 100 complex i particles from eight image pairs , recorded with a relative tilt angle of 10 , were extracted and subjected to tilt - pair analysis with frealign . the outer radius of the plot is 40 and the orange circle centered at the expected tilt angle has a radius of 6. phases that are beyond 10  in each of the micrographs used in the final data set ( frames 1 - 32 ) were randomised , and then refinement was performed as for a normal data set ( fsc summed image corresponding to frames 1 - 32 ) . as expected , the graph shows a drop in the fourier shell correlation ( fsc ) curve at 10  , validating the presence of information beyond 10  in the images . note that the use of gold - standard refinement procedures in relion prevents any overfitting , and this test was done only as an additional control . d ) fsc curves of the final map and of the model versus the map . the curve in red is the gold - standard fsc of the final map ( after classification ) and the resolution at fsc = 0.143 is ~4.95  . the curve in cyan is the fsc between the final map and the model , and at fsc = 0.5 the resolution is 6.7  . note that the present model is not complete since it is only a polyalanine model without any side chains , and loop regions in a number of subunits have not been modelled . the left - hand panel ( with lower density threshold ) shows that the detergent / phospholipid belt is of lower resolution , and the protein regions of the map show resolution distributed from 5 to 6  . in the right - hand panel the map is shown at higher density threshold , so the detergent / phospholipid belt is not visualised . some of the interior parts of the map have resolution of 4.8 - 5  . a ) nd2 is shown from the membrane plane , high - lighting the densities for three aromatic sidechains and one of the helix - breaking loops . c ) the density for a [ 4fe-4s ] cluster and surrounding protein is shown in the psst subunit . d ) a region of the 49 kda subunit shows a well resolved -helical stretch and aromatic side - chains , and the -strands are beginning to be resolved . e ) subunit b8 is an example of a supernumerary subunit in a peripheral region of the molecule . f ) in the 39 kda subunit density consistent with a bound nucleotide is observed , in a similar position to in homologous structures , and as expected from analysis of y. lipolytica complex i. however , the present resolution of the map precludes the inclusion of this nucleotide in the final model . the core subunits from b. taurus are in blue , and from t. thermophilus ( 4hea.pdb ) in orange . top : the nd2 , nd4 and nd5 domain is rotated in b. taurus relative to in t. thermophilus , increasing the curvature in the b. taurus membrane domain . the complex is viewed along the 11 rotation vector ( orange ) that maps the t. thermophilus nd2 , nd4 and nd5 domain to the b. taurus domain , along with a small 5  translation to superimpose the domain centres . correspondingly , the nd3 , nd4l and nd6 domains are superimposed by a 4 rotation and a 1  translation . rotation of nd2 , 4 and 5 about the long axis of the domain , as noted for y. lipolytica , is not observed . bottom : the nadh dehydrogenase domain containing the 51 and 24 kda subunits is rotated by 23 and translated by 14  in b. taurus , relative to in t. thermophilus , causing the fes chains to diverge as the distance from nd1 increases . a similar rotation was observed in y. lipolytica . the complex is viewed from behind nd1 . correspondingly , the 49 kda , psst and tyky subunits are superimposed by a 6 rotation and a 2  translation . the structures were analysed using superpose from the ccp4 suite and the 75 kda and 30 kda subunits were not included due to their lower structural conservation . a ) the structure of each subunit from t. thermophilus ( wheat , 4hea.pdb ) has been superimposed separately on its corresponding subunit from b. taurus ( coloured as labelled ) with the transverse helix plus tmh16 of nd5 also aligned separately . the complexes are viewed from behind nd1 ( top ) , from the side ( middle ) and from the matrix ( bottom , nd subunits only ) . b ) observed differences in the structures of the core subunits of b. taurus and t. thermophilus complexes i. grey , conserved structure from b. taurus and t. thermophilus ( 4hea.pdb ) ; red , structural elements present only in t. thermophilus ; blue , structural elements present only in b. taurus . the c - terminal domain of the 75 kda subunit is not resolved in b. taurus , but its structure is clearly different to in t. thermophilus . table 1reference table for the nomenclature of the core subunits of complex iin the text the names of the subunits from b. taurus are used , with the names from the human enzyme presented alongside as appropriate.domainchain identifier 
 bos taurus 

 homo sapiens 

 yarrowia lipolytica 

 thermus thermophilics 

 escherichia coli 
 hydrophilic domaing75 kdandufs1nuamnqo3nuogf51 kdandufv1nubmnqo1nuofd49 kdandufs2nucmnqo4nuocdc30 kdandufs3nugmnqo5e24 kdandufv2nuhmnqo2nuoebpsstndufs7nukmnqo6nuobitykyndufs8nuimnqo9nuol 

 membrane domainhnd1nd1nu1mnqo8nuohnnd2nd2nu2mnqo14nuonand3nd3nu3mnqo7nuoamnd4nd4nu4mnqo13nuomlnd5nd5nu5mnqo12nuoljnd6nd6nu6mnqo10nuojknd4lnd4lnulmnqo11nuok in the text the names of the subunits from b. taurus are used , with the names from the human enzyme presented alongside as appropriate . table 2summary of the models of the core subunits of b. taurus complex isubunittotal residues*modelled residuespoorly resolved / uncertain residue numberingunresolved residuesunresolved elements ( > 10 residues)%modelled%identicalrmsdnd13183 - 200219 - 242253 - 3151 - 2201 - 218243 - 252316 - 318matrix loop ( tmh 5 - 6)ims loop ( tmh 6 - 7)90%(285/318)42%(132/318)1.60 nd23472 - 300320 - 346tmh111301 - 319347matrix loop ( tmh 10 - 11)94%(326/347)25%(86/347)2.08 nd31152 - 2352 - 112124 - 51113 - 115matrix loop ( tmh 1 - 2)72%(83/115)27%(31/115)2.05 nd44593 - 415430 - 455tmh141 - 2416 - 429456 - 459matrix loop ( tmh 13 - 14)96%(439/459)24%(111/459)2.20 nd4l981 - 8485 - 98matrix loop ( c - terminus)86%(84/98)21%(21/98)2.66 nd56064 - 2228 - 358363 - 400408 - 466487 - 513520 - 604tmh1tmh13 & tmh14tmh15transverse helix & tmh161 - 323 - 27359 - 362401 - 407467 - 486514 - 519605 - 606matrix loop ( tmh 1 - 2)matrix loop ( tmh 11 - 12)ims loop ( tmh 12 - 13)ims loop ( tmh 14 - 15)tmh 15 to transverse helix92%(558/606)31%(187/606)2.53 nd61752 - 7685 - 107140 - 172tmh5177 - 84108 - 139173 - 175matrix loop ( tmh 3 - 4)ims loop ( tmh 4 - 5)75% ( 131/175)16% ( 28/175)1.83  

 75 kdandufs17048 - 125136 - 318326 - 347367 - 400404 - 410425 - 495525 - 530542 - 627the large domain ( 222 - 704 ) is generally poorly resolved.the sequence alignment isweak and the secondary structurecontent low . residues 404 - 629are particularly poorly resolved.1 - 7126 - 135319 - 325348 - 366400 - 403411 -424496 - 524531 -541628 - 704probable loop regionprobable loop regionprobable loop regionprobable loop regionprobable subdomain75% ( 527/704)1 - 221 : 92%222 - 704 : 67%27% ( 189/704)1 - 221 40%222 - 704 : 21%1.96 1 - 221 : 1.57 222 - 704 : 2.11 51 kdandufv144431 - 441flavin and nadh binding site ( 63 - 72 , 99 - 104 , 181 - 189 , 300 - 304 , 327 - 333)1 - 30442 - 444n - terminal peptide93% ( 411/444)43% ( 191/444)1.61 49 kdandufs243047 - 4303-strand -sheet ( 47 - 79)1 - 46n - terminal region89% ( 384/430)42% ( 179/430)1.41 30 kdandufs322815 - 168numbering uncertain to 72loop / -strand ( 73 - 83)1 - 14169 - 228n - terminal peptidec - terminal region68% ( 154/228)24% ( 54/228)1.66 24 kdandufv221720 - 178loop 126 - 1321 - 19179 - 217n - terminal peptidec - terminal region73% ( 159/217)27% ( 59/217)1.57 psstndufs717927 - 169loop 68 - 791 - 26170 - 179n - terminal peptide80% ( 143/179)49% ( 88/179)1.44 tykyndufs817615 - 1761 - 14n - terminal peptide92% ( 162/176)36% ( 63/176)1.89 *for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein,.the % identity and the rmsd ( root mean square deviation calculated using pdbefold ) are between the sequences and structures of the subunits of b. taurus and t. thermophilus ( 4hea.pdb ) complexes i. for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein , . the % identity and the rmsd ( root mean square deviation calculated using pdbefold ) are between the sequences and structures of the subunits of b. taurus and t. thermophilus ( 4hea.pdb ) complexes i. table 3distances between the redox cofactors in structural models of complex i 
 t. thermophilus 

 b. taurus 


 hydrophilic domain ( 2fug.pdb)complex i ( 4hea.pdb)complex i ( this work ) 

 cofactors*centreedgecentreedgecentreedgen1a - flavin15.412.315.913.115.913.1flavin - cluster 1 ( n3)12.57.612.27.312.27.2n1a - cluster 1 ( n3)22.119.422.319.721.118.0cluster 1 ( n3 ) - cluster 214.011.013.710.714.011.0cluster 1 ( n3 ) - cluster 317.413.817.113.418.414.5cluster 2 - cluster 313.510.713.09.912.79.7cluster 3 - cluster 412.28.512.48.612.88.7cluster 4 - cluster 516.814.016.513.616.814.0cluster 5 - cluster 612.19.412.19.312.19.3cluster 6 - cluster 7 ( n2)13.710.513.510.213.610.5cluster 1 ( n3 ) - cluster 7 ( n2)61.157.660.557.061.558.1*the [ 2fe-2s ] cluster in the 24 kda subunit ( known as n1a ) is on the other side of the flavin from the main cofactor chain . the [ 4fe-4s ] cluster in the 51 kda subunit ( known as n3 ) is the first cluster in the chain and the [ 4fe-4s ] cluster in subunit psst ( known as n2 ) is the last ( seventh ) cluster in the chain.the distances are in ngstroms , between the geometric centres of the fe and s cluster cores or the flavin isoalloxazine ring system ( centre ) , or between the centres of the two closest atoms ( edge ) as commonly used in calculations of electron transfer rates . distances are estimated to be accurate to within 1 .the position of the flavin in b. taurus is poorly resolved and has been approximated using its position in 4hea.pdb . the [ 2fe-2s ] cluster in the 24 kda subunit ( known as n1a ) is on the other side of the flavin from the main cofactor chain . the [ 4fe-4s ] cluster in the 51 kda subunit ( known as n3 ) is the first cluster in the chain and the [ 4fe-4s ] cluster in subunit psst ( known as n2 ) is the last ( seventh ) cluster in the chain . the distances are in ngstroms , between the geometric centres of the fe and s cluster cores or the flavin isoalloxazine ring system ( centre ) , or between the centres of the two closest atoms ( edge ) as commonly used in calculations of electron transfer rates . the position of the flavin in b. taurus is poorly resolved and has been approximated using its position in 4hea.pdb . sapiens subunit*subcomplexsequence informationpredicted tmhs10 kdandufv3i and i018 kdandufs4i and i015 kdandufs5i onlycx9c motif , intermembrane space013 kdandufs6i and ipfam zinc - finger motif cx8hx15cx2c0mwfendufa1i only1b8ndufa2i and i0b9ndufa3i only1b13ndufa5i and i0b14ndufa6i onlylyr motif0b14.5andufa7i and i0pgivndufa8i onlytwo cx9c motifs , pfam chch domain intermembrane space039 kdandufa9i onlyshort - chain dehydrogenase reductase family , nadp binding,042 kdandufa10i only ( low level)similarity to deoxynucleoside kinases0b14.7ndufa11i ( i at low level)3 or 4b17.2ndufa12i and i0b16.6ndufa13i and i1sdapndufab1both i iacyl - carrier protein,0mnllndufb1i0 ( or 1)agggndufb2i1 ( or 0)b12ndufb3i1b15ndufb4both i and i1sgdhndufb5i1b17ndufb6i1b18ndufb7icx9c motif , intermembrane space0ashindufb8i1b22ndufb9ilyr motif0pdswndufb10i0esssndufb11i1kfyindufc1none1b14.5bndufc2i ( low level)1 or 2*the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i.subcomplex i , which contains the seven hydrophilic core subunits and eight to nine supernumerary subunits , is considered to represent a significant portion of the hydrophilic domain of complex i. subcomplex i , which contains all the subunits of subcomplex i plus core subunit nd6 and nine to ten additional supernumerary subunits , represents the hydrophilic domain of complex i plus associated membrane subunits . subcomplex i , which contains nd4 and nd5 and 12 to 13 supernumerary subunits , represents part of the membrane domain.tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) and are presented as consensus values with less represented values in brackets and single outliers discarded . the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i. subcomplex i , which contains the seven hydrophilic core subunits and eight to nine supernumerary subunits , is considered to represent a significant portion of the hydrophilic domain of complex i. subcomplex i , which contains all the subunits of subcomplex i plus core subunit nd6 and nine to ten additional supernumerary subunits , represents the hydrophilic domain of complex i plus associated membrane subunits . subcomplex i , which contains nd4 and nd5 and 12 to 13 supernumerary subunits , represents part of the membrane domain . tmhs were predicted using tmhmm2 , hmmtop2 and the topcons suite ( seven methods in total ) and are presented as consensus values with less represented values in brackets and single outliers discarded . table 5summary of the models of the supernumerary subunits of b. tauruscomplex isubunitchain identifiertotal residues*pdb modelaligned residues%identicalmodelled residues%modelledrmsd42 kdandufa10o3202ocp21 - 25221% ( 49/232)22 - 5479 - 167172 - 210222 - 24157% ( 181/320)1.91 39 kdandufa9p3452q1w19 -32513% ( 41/307)19 - 185203 - 250285 - 32173% ( 252/345)2.52 18 kdandufs4q1332jya33 - 13337% ( 37/101)33 - 5976 - 11652% ( 69/133)2.42 13 kdandufs6r962jrr44 - 9634% ( 18/53)47 - 9349% ( 47/96)1.97 b8ndufa2s991s3a1 - 9994% ( 93/99)17 - 9681% ( 80/99)2.18 sdap-ndufab1t881f808 - 8436% ( 28/77)9 - 2328 - 8281% ( 71/88)1.18 sdap-ndufab1u881f808 - 8436% ( 28/77)8 - 8285% ( 75/88)1.36 b13ndufa5v1161 - 7161% ( 71/116)b14ndufa6w1281 - 7256% ( 72/128)pgivndufa8x1722lql35 - 11423% ( 18/80)1 - 8046% ( 79/172)2.40 b14.7ndufa11y1411 - 10675% ( 106/141)b16.6ndufa13z14433 - 9745% ( 65/144)b9ndufa3 or mwfendufa1a1541 - 2946% ( 71/154)b1 - 42*for proteins with a mitochondrial - targeting pre - sequence , residue 1 is the first residue of the mature protein 3.known structures with high homology to the complex i subunits were identified by hhpred.rmsd : root mean square deviation calculated using pdbefold between the structures of the subunits of b. taurus complex i and the pdb model structures.subunit with less certain assignment.residue numbers are arbitrary and not assigned to the sequence . for proteins with a mitochondrial - targeting pre - sequence , rmsd : root mean square deviation calculated using pdbefold between the structures of the subunits of b. taurus complex i and the pdb model structures .
complex i ( nadh : ubiquinone oxidoreductase ) is essential for oxidative phosphorylation in mammalian mitochondria . 
 it couples electron transfer from nadh to ubiquinone with proton translocation across the energy - transducing inner membrane , providing electrons for respiration and driving atp synthesis . 
 mammalian complex i contains 44 different nuclear- and mitochondrial - encoded subunits , with a combined mass of 1 mda . 
 the fourteen conserved 
 core subunits have been structurally defined in the minimal , bacterial complex , but the structures and arrangement of the 30  supernumerary subunits are unknown . here , we describe a 5  resolution structure of complex i from bos taurus heart mitochondria , a close relative of the human enzyme , determined by single - particle electron cryo - microscopy . 
 we present the structures of the mammalian core subunits that contain eight iron - sulphur clusters and 60 transmembrane helices , identify 18 supernumerary transmembrane helices , and assign and model 14 supernumerary subunits . 
 thus , we significantly advance knowledge of the structure of mammalian complex i and the architecture of its supernumerary ensemble around the core domains . 
 our structure provides insights into the roles of the supernumerary subunits in regulation , assembly and homeostasis , and a basis for understanding the effects of mutations that cause a diverse range of human diseases .