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 7-cyclohexyl-1-heptyl--d - maltoside ( cymal 7 ) , as described previously10,31 .
images were recorded using a 300 kev fei titan krios electron microscope with epu software , with the specimen temperature at -186 c . a falcon ii cmos ( complementary metal oxide semiconductor ) direct electron detector was used for imaging at 105,263x magnification ( nominally 59,000x ) , corresponding to a sampling density of 1.33  pixel , at 2.4 - 4.2 m under - focus in 0.3 m steps , with the autofocus routine performed every 8 m to give a range of defocus .
each image was exposed for 2 s ( total dose ~35 e  ) and an in - house protocol was used to capture 34 movie frames .
the frames were aligned using unblur ( without dose filtering)32 and the ctf was determined with ctffind433 .
a total of 139,456 particles were picked manually and extracted using a box of 360 pixels .
the 5  resolution map described previously10 was low - pass filtered to 60  and used as the reference map .
the whole data set was subjected to the auto - refine routine in relion , followed by modelling of the beam - induced movement ( using a running average of 7 frames ) and b - factor weighting .
all the resolutions described here are defined at fsc = 0.143 following application of a shape mask , phase randomisation to check for effects of the mask , and correction for the modulation transfer function of the detector .
the resolution of the data set containing all the particles after b - factor weighting and refinement was 4.16  , with an estimated orientation accuracy of 0.93 degrees .
the b - factor weighted particles were subjected to 3d classification into eight classes using an angular sampling of 0.9 for 25 iterations , with the resolution limited to 8  .
three major classes were identified , containing 48,033 ( class 1 ) , 33,301 ( class 2 ) and 19,306 ( class 3 ) particles .
each class was refined individually , providing maps with overall resolution of 4.27  for class 1 , 4.35  for class 2 and 5.6  for class 3 .
the maps were sharpened with b - factors of -114 for class 1 , -110 for class 2 and -125 for class 3 .
local analysis of the resolution was performed using resmap35 ( ed figures 1 and 2 ) .
note that the map used as the reference for refinement is a class 1 map , which we described previously as the major class10 , and that the number of particles required to achieve the reported resolutions indicates the need for future improvements in both the biochemical homogeneity and specimen preparation of the samples .
the first model was built using the map from the complete dataset , with cross - referencing to the maps from classes 1 and 2 , using the 5  model for b. taurus complex i described previously ( 4uq8.pdb10 ) as the initial template .
this unrefined polyalanine model contains models for the fourteen core subunits that are structural homologues of the subunits of the bacterial enzyme9 , partial models for fourteen assigned supernumerary subunits , and a further 21 polypeptide chains from unassigned supernumerary structures .
assignments to some of these chains were subsequently proposed for a further eight supernumerary subunits using the 6.8  x - ray crystallographic structure of subcomplex i from b. taurus complex i12 .
the new maps show clear connectivity within the density features , allowing many of the previously traced chains to be extended significantly , and some of them to be joined together .
furthermore , the helical pitches of most of the tmhs and of many of the helices in the globular subunits are now clear , and the -strands are well separated .
these significant improvements in the density , together with information from secondary structure analyses and homologous structures , allowed improved and more complete models to be built for all 45 subunits ( see below ) .
note that the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i37 and that there are two copies of subunit sdap10 .
the only substantial unmodelled protein densities are underneath the tip of the membrane domain and are accounted for by the termini of two supernumerary subunits ( b18 and ashi ) . in well - resolved regions of the map , protruding densities of the side chains of the bulky aromatic residues phe , tyr and trp , along with some side chain densities from arg and his , are clearly visible ( ed figures 3 and 4 ) . for those subunits which had already been assigned these side chain features
side chains were added in well - resolved regions , but omitted when their density features are unclear .
the assignments of four subunits that were previously assigned in pairs ( b9 and mwfe10 , and pdsw and b1812 ) were also confirmed . for three subunits ( b8 , sdap- and sdap- ) models of the human homologues
are available in the pdb ( 1s3a.pdb38 and 2dnw.pdb ) and were used to assign the residues .
for highly - conserved regions of the 51 kda , 24 kda ( ndufv2 ) and 75 kda subunits , residue assignments were supported by the structure of complex i from t. thermophilus9 . in some less well - resolved regions of the map it is not possible to assign the sequence confidently using the current data . in these regions
the polyalanine model has been retained ( residue names unk ) , but the residues for each subunit have been numbered as accurately as possible , to provide a guide to the location of individual residues .
the exceptions are subunits b12 , 10 kda and part of b14.5a , for which residue numbers can not be confidently proposed . in summary ,
the models described are mixed models in which the residues in some subunits are fully assigned , some partly assigned , and others not assigned at all ( ed tables 1 and 2 ) .
the patterns of the bulky residues observed in the tmh - containing regions of unknown densities were compared with the amino acid sequences of candidate subunits . by combining this information with information from secondary structure analyses , supported with biochemical knowledge ,
the seven hitherto - unassigned tmhs were assigned and the subunits modelled as described above . subunits b12 and aggg were assigned to the two tmhs on the tip of the membrane domain , but lack of clear features in the densities means that our specific assignment of b12 to chain k and aggg to chain j is less confident , so we can not exclude the possibility that they have been reversed .
the two additional tmh - like densities observed in the structure of subcomplex i12 are clearly absent from the cryoem maps , so they are attributed to an artefact produced by crystal contacts with either dissociated subunits or the reorganised transverse helix .
remaining polypeptide chains , located on the outside of the hydrophilic domain and the ims face of the membrane domain , were assigned by combining secondary structure analyses with biochemical knowledge and by using the densities from bulky residues , and the residues and side chains assigned and modelled where possible .
maps with different b - factor sharpening were used to help with chain tracing and assignment of residues , and the model was checked for consistency with the individual maps from the three different classes .
the geometry of the model was improved by cycles of manual adjustment in coot36 , real - space refinement by phenix39 and refinement by refmac40 , with secondary structure restraints .
separate models were subsequently created for classes 1 and 2 by rigid - body fitting of each subunit , manual adjustment to account for significant local differences identified in the density maps , and cycles of adjustment and refinement as described above . to provide some reassurance of the refinement , the coordinates for classes 1 and 2 were shifted by 0.1  and the b - factors re - set to 75 , then the coordinates were refined against one of the half maps .
the resulting models were then used to calculate fourier - shell correlation ( fsc ) curves for both half maps ; as shown in ed figure 2 the curves display very little evidence of overfitting .
the class 3 model was created from the class 1 model by rigid - body fitting .
it was created purely for comparison with classes 1 and 2 so the individual subunit models were retained unchanged from class 1 , the model was not deleted in the regions of poor density reported in ed figure 5 , and no refinement was performed .
the sequences of all the subunits are numbered starting from residue 1 of the mature proteins2 .
the naming of the chains has been retained as much as possible from our previous model ( 4uq8.pdb ) .
the names are unchanged for a to z and a to n , except that the previous chains d and e have been combined to form new chain d ( b14.5b ) and chain e has been reallocated ( 15 kda ) .
previous chains o to w all represented sections of subunits that have now been combined ; new chains n to s have been reallocated .
after modelling the protein , we observed two additional short , elongated densities located at interfaces between core membrane - domain subunits , which may represent phospholipid molecules .
they are between nd2-tmh10 ( residues 291 - 295 ) , nd4-tmh5 ( residues 144 - 147 ) and the nd5 transverse helix ( residues 564 - 567 ) , and between nd4-tmh11 ( residues 356 - 360 ) and nd5-tmh4 ( residues 116 - 123 ) .
these densities have not been modelled , because similar densities are observed elsewhere within the detergent / phospholipid belt but at lower contour levels , so we can not exclude the possibility that they are due to noise in the density map at the current resolution .
the identification of tmhs in the sequences and the structures of homologous proteins were described previously10 .
the subunit interactions in ed table 3 were calculated with ncont in ccp444 , and defined as a centre - to - centre distance of less than 5  between any two atoms . for some subunits , such as b8 and sdap- ,
the interactions are limited to one subunit , while other subunits with long loops and extended structures , such as sgdh , form multiple interactions .
some of the residues in our current model lack side chains so the number of interactions detected may increase in future models .
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 7-cyclohexyl-1-heptyl--d - maltoside ( cymal 7 ) , as described previously10,31 .
images were recorded using a 300 kev fei titan krios electron microscope with epu software , with the specimen temperature at -186 c . a falcon ii cmos ( complementary metal oxide semiconductor ) direct electron detector was used for imaging at 105,263x magnification ( nominally 59,000x ) , corresponding to a sampling density of 1.33  pixel , at 2.4 - 4.2 m under - focus in 0.3 m steps , with the autofocus routine performed every 8 m to give a range of defocus . each image was exposed for 2 s ( total dose ~35 e  ) and an in - house protocol was used to capture 34 movie frames .
the frames were aligned using unblur ( without dose filtering)32 and the ctf was determined with ctffind433 .
a total of 139,456 particles were picked manually and extracted using a box of 360 pixels .
the 5  resolution map described previously10 was low - pass filtered to 60  and used as the reference map .
the whole data set was subjected to the auto - refine routine in relion , followed by modelling of the beam - induced movement ( using a running average of 7 frames ) and b - factor weighting .
all the resolutions described here are defined at fsc = 0.143 following application of a shape mask , phase randomisation to check for effects of the mask , and correction for the modulation transfer function of the detector .
the resolution of the data set containing all the particles after b - factor weighting and refinement was 4.16  , with an estimated orientation accuracy of 0.93 degrees .
the b - factor weighted particles were subjected to 3d classification into eight classes using an angular sampling of 0.9 for 25 iterations , with the resolution limited to 8  .
three major classes were identified , containing 48,033 ( class 1 ) , 33,301 ( class 2 ) and 19,306 ( class 3 ) particles .
each class was refined individually , providing maps with overall resolution of 4.27  for class 1 , 4.35  for class 2 and 5.6  for class 3 .
the maps were sharpened with b - factors of -114 for class 1 , -110 for class 2 and -125 for class 3 .
local analysis of the resolution was performed using resmap35 ( ed figures 1 and 2 ) .
note that the map used as the reference for refinement is a class 1 map , which we described previously as the major class10 , and that the number of particles required to achieve the reported resolutions indicates the need for future improvements in both the biochemical homogeneity and specimen preparation of the samples .
the first model was built using the map from the complete dataset , with cross - referencing to the maps from classes 1 and 2 , using the 5  model for b. taurus complex i described previously ( 4uq8.pdb10 ) as the initial template .
this unrefined polyalanine model contains models for the fourteen core subunits that are structural homologues of the subunits of the bacterial enzyme9 , partial models for fourteen assigned supernumerary subunits , and a further 21 polypeptide chains from unassigned supernumerary structures .
assignments to some of these chains were subsequently proposed for a further eight supernumerary subunits using the 6.8  x - ray crystallographic structure of subcomplex i from b. taurus complex i12 .
the new maps show clear connectivity within the density features , allowing many of the previously traced chains to be extended significantly , and some of them to be joined together .
furthermore , the helical pitches of most of the tmhs and of many of the helices in the globular subunits are now clear , and the -strands are well separated .
these significant improvements in the density , together with information from secondary structure analyses and homologous structures , allowed improved and more complete models to be built for all 45 subunits ( see below ) .
note that the former subunit mlrq ( ndufa4 ) is no longer considered a subunit of complex i37 and that there are two copies of subunit sdap10 .
the only substantial unmodelled protein densities are underneath the tip of the membrane domain and are accounted for by the termini of two supernumerary subunits ( b18 and ashi ) . in well - resolved regions of the map , protruding densities of the side chains of the bulky aromatic residues phe , tyr and trp , along with some side chain densities from arg and his , are clearly visible ( ed figures 3 and 4 ) . for those subunits which had already been assigned these side chain features
side chains were added in well - resolved regions , but omitted when their density features are unclear .
the assignments of four subunits that were previously assigned in pairs ( b9 and mwfe10 , and pdsw and b1812 ) were also confirmed . for three subunits ( b8 , sdap- and sdap- ) models of the human homologues are available in the pdb ( 1s3a.pdb38 and 2dnw.pdb ) and were used to assign the residues . for highly - conserved regions of the 51 kda ,
24 kda ( ndufv2 ) and 75 kda subunits , residue assignments were supported by the structure of complex i from t. thermophilus9 . in some less well - resolved regions of the map it is not possible to assign the sequence confidently using the current data . in these regions
the polyalanine model has been retained ( residue names unk ) , but the residues for each subunit have been numbered as accurately as possible , to provide a guide to the location of individual residues .
the exceptions are subunits b12 , 10 kda and part of b14.5a , for which residue numbers can not be confidently proposed . in summary ,
the models described are mixed models in which the residues in some subunits are fully assigned , some partly assigned , and others not assigned at all ( ed tables 1 and 2 ) .
the patterns of the bulky residues observed in the tmh - containing regions of unknown densities were compared with the amino acid sequences of candidate subunits . by combining this information with information from secondary structure analyses , supported with biochemical knowledge ,
the seven hitherto - unassigned tmhs were assigned and the subunits modelled as described above . subunits b12 and aggg were assigned to the two tmhs on the tip of the membrane domain , but lack of clear features in the densities means that our specific assignment of b12 to chain k and aggg to chain j is less confident , so we can not exclude the possibility that they have been reversed .
the two additional tmh - like densities observed in the structure of subcomplex i12 are clearly absent from the cryoem maps , so they are attributed to an artefact produced by crystal contacts with either dissociated subunits or the reorganised transverse helix .
remaining polypeptide chains , located on the outside of the hydrophilic domain and the ims face of the membrane domain , were assigned by combining secondary structure analyses with biochemical knowledge and by using the densities from bulky residues , and the residues and side chains assigned and modelled where possible . maps with different b - factor sharpening were used to help with chain tracing and assignment of residues , and the model was checked for consistency with the individual maps from the three different classes .
the geometry of the model was improved by cycles of manual adjustment in coot36 , real - space refinement by phenix39 and refinement by refmac40 , with secondary structure restraints .
separate models were subsequently created for classes 1 and 2 by rigid - body fitting of each subunit , manual adjustment to account for significant local differences identified in the density maps , and cycles of adjustment and refinement as described above . to provide some reassurance of the refinement , the coordinates for classes 1 and 2 were shifted by 0.1  and the b - factors re - set to 75 , then the coordinates were refined against one of the half maps .
the resulting models were then used to calculate fourier - shell correlation ( fsc ) curves for both half maps ; as shown in ed figure 2 the curves display very little evidence of overfitting .
the class 3 model was created from the class 1 model by rigid - body fitting .
it was created purely for comparison with classes 1 and 2 so the individual subunit models were retained unchanged from class 1 , the model was not deleted in the regions of poor density reported in ed figure 5 , and no refinement was performed .
the sequences of all the subunits are numbered starting from residue 1 of the mature proteins2 .
the naming of the chains has been retained as much as possible from our previous model ( 4uq8.pdb ) .
the names are unchanged for a to z and a to n , except that the previous chains d and e have been combined to form new chain d ( b14.5b ) and chain e has been reallocated ( 15 kda ) .
previous chains o to w all represented sections of subunits that have now been combined ; new chains n to s have been reallocated .
after modelling the protein , we observed two additional short , elongated densities located at interfaces between core membrane - domain subunits , which may represent phospholipid molecules .
they are between nd2-tmh10 ( residues 291 - 295 ) , nd4-tmh5 ( residues 144 - 147 ) and the nd5 transverse helix ( residues 564 - 567 ) , and between nd4-tmh11 ( residues 356 - 360 ) and nd5-tmh4 ( residues 116 - 123 ) .
these densities have not been modelled , because similar densities are observed elsewhere within the detergent / phospholipid belt but at lower contour levels , so we can not exclude the possibility that they are due to noise in the density map at the current resolution .
the identification of tmhs in the sequences and the structures of homologous proteins were described previously10 . cavities and channels in the structures
the subunit interactions in ed table 3 were calculated with ncont in ccp444 , and defined as a centre - to - centre distance of less than 5  between any two atoms .
for some subunits , such as b8 and sdap- , the interactions are limited to one subunit , while other subunits with long loops and extended structures , such as sgdh , form multiple interactions .
some of the residues in our current model lack side chains so the number of interactions detected may increase in future models .
a ) the map , shown at two different contour levels , is coloured according to the local resolution , as determined by resmap35 . at the higher contour level ( left )
, the majority of the protein is resolved to 3.94.7  ; only the very peripheral regions ( parts of the 51 kda , 24 kda and 75 kda subunits in the matrix arm , and the distal end of the membrane arm ) are at lower resolutions of 56  . at the lower contour level ( right ) , the detergent / lipid belt dominates the 79  resolution range .
b ) a slice through the map shows that large portions of the central , core regions are resolved to 4  or better .
c ) the fsc curve defines an estimated overall resolution of 4.16  at fsc = 0.143 .
panels a , b and c show data on classes 1 , 2 and 3 , respectively .
classes 1 and 2 display similar distributions in local resolution , with the majority of the protein in the range 45  . in class 3 the majority of protein
the refined models agree very well with the maps as shown by comparison of the fsc curves ( red ) and the fsc curves from the half - maps ( blue ) , and the similarity of the resolution values at fsc 0.143 and 0.5
. the estimated resolutions , defined where the line at fsc = 0.143 crosses the blue curve , are 4.27  for class 1 , 4.35  for class 2 and 5.6  for class 3 .
cross - validation of the refinement parameters , confirming lack of over - fitting . for classes 1 and 2 , one of the half maps was used for refinement then the fsc curves were calculated for each of the two half maps using the same model .
a ) a selection of tmhs from the membrane domain : tmh3 from nd1 , the distorted tmhs in nd6 , nd4 and nd5 , and a discontinuous tmh from nd2 .
the series of tmhs from left to right illustrates the decrease in resolution along the domain .
b ) the two [ 4fes4 ] clusters in the 75 kda subunit ( density in red , at higher contour level ) with the protein ligating one of them .
b ) subunit b14.5 , containing two tmhs . the n- and c - terminal loops are not shown .
c ) 15 kda subunit on the ims face , containing a chch domain with two disulphide bonds . the n- and
d ) the seven - strand -sheet in the 39 kda subunit , showing the separation of the strands , and the bound nucleotide ( red density ) modelled as nadph .
e ) helix 1 , one of the arginine - rich helices , in b22 , and sdap- , on the matrix side of the tip of the membrane domain .
inset : the weak density attached to ser44 in sdap- attributed to the attached pantetheine-4'-phosphate group ( side chain of ser44 not shown ) .
a ) the structures for classes 1 and 3 have been superimposed using nd1 and nd3 . in class 3 ,
relative to class 1 , the hydrophilic and distal membrane domains are both rotated and shifted , but the change in the membrane domain dominates .
although the changes appear to make the angle of the l - shaped molecule increase they do not originate from a hinge - like motion at the interface of the hydrophilic and membrane domains .
class 1 is in red , class 3 is in red ( nd1 domain ) , wheat , blue and cyan .
details of the composition and movement of the domains are given in ed table 4 .
b ) the density for class 3 ( white ) is presented with the model for well - resolved regions of class 3 in blue ( the model is enclosed in the density ) and the model for poorly resolved regions in red ( the model appears outside the density ) .
the poorly resolved regions include the n - terminus of 49 kda and the transverse helix in nd5 , as well as elements of nd4 , nd6 , b14.7 , esss and b15 .
the names of the human , t. thermophilus and e. coli subunits ( if different to the names in b. taurus ) . the assignments of b12 and aggg may be reversed arbitrary residue numbers the n - terminus of 49 kda ( residues 5 - 39 ) is displaced in class 1 relative to class 2 when considered from the core fold of the subunit because it lies on the surface of the membrane domain and moves with nd2 .
 complex i ( nadh : ubiquinone oxidoreductase ) , one of the largest membrane - bound enzymes in the cell , powers atp synthesis in mammalian mitochondria by using the reducing potential of nadh to drive protons across the inner membrane . 
 mammalian complex i1 contains 45 subunits , comprising 14 core subunits that house the catalytic machinery and are conserved from bacteria to humans , and a mammalian - specific cohort of 31 supernumerary subunits1,2 . 
 knowledge about the structures and functions of the supernumerary subunits is fragmentary . here , we describe a 4.2  resolution single - particle cryoem structure of complex i from bos taurus . 
 we locate and model all 45 subunits to provide the entire structure of the mammalian complex . 
 furthermore , computational sorting of the particles identified different structural classes , related by subtle domain movements , which reveal conformationally - dynamic regions and match biochemical descriptions of the  active - to - deactive enzyme transition that occurs during hypoxia3,4 . 
 thus , our structures provide a foundation for understanding complex i assembly5 and the effects of mutations that cause clinically - relevant complex i dysfunctions6 , insights into the structural and functional roles of the supernumerary subunits , and new information on the mechanism and regulation of catalysis . 
Methods
Protein preparation
CryoEM specimen preparation, imaging and image processing
Model building and analysis
Bioinformatics
Extended Data