the  and  subunits of pcc were co - expressed in e. coli , with a his - tag on the  subunit .
crystals were obtained by the microbatch method under oil , and the structures were determined by the molecular replacement method .
frozen hydrated human pcc particles at 70 g / ml concentration were imaged at 50,000 magnification in a 100 kv cryo - electron microscope .
a featureless gaussian oval was used to obtain a low resolution ( 40  ) model from negative stain electron microscope images .
a 15  resolution 3d reconstruction was obtained from ~10,000 cryo - em particle images , using the structure from the negative stain images as the initial model .
site - specific and deletion mutants were designed based on the structural information , and their effects on the formation of the holoenzyme was assessed by nickel affinity chromatography .
the catalytic activity of pcc was determined by a coupled enzyme assay , monitoring the hydrolysis of atp .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
the  and  subunits of pcc were co - expressed in e. coli , with a his - tag on the  subunit .
crystals were obtained by the microbatch method under oil , and the structures were determined by the molecular replacement method .
frozen hydrated human pcc particles at 70 g / ml concentration were imaged at 50,000 magnification in a 100 kv cryo - electron microscope .
a featureless gaussian oval was used to obtain a low resolution ( 40  ) model from negative stain electron microscope images .
a 15  resolution 3d reconstruction was obtained from ~10,000 cryo - em particle images , using the structure from the negative stain images as the initial model .
site - specific and deletion mutants were designed based on the structural information , and their effects on the formation of the holoenzyme was assessed by nickel affinity chromatography .
the catalytic activity of pcc was determined by a coupled enzyme assay , monitoring the hydrolysis of atp .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
 propionyl - coenzyme a carboxylase ( pcc ) , a mitochondrial biotin - dependent enzyme , is essential for the catabolism of the amino acids thr , val , ile and met , cholesterol , and fatty acids with an odd number of carbon atoms . 
 deficiencies of pcc activity in humans are linked to the disease propionic acidemia ( pa ) , an autosomal recessive disorder that can be fatal in infants 14 . 
 the holoenzyme of pcc is an 66 dodecamer , with a molecular weight of 750 kd . 
 the  subunit contains the biotin carboxylase ( bc ) and biotin carboxyl carrier protein ( bccp ) domains , while the  subunit supplies the carboxyltransferase ( ct ) activity . here 
 we report the crystal structure at 3.2  resolution of a bacterial pcc 66 holoenzyme as well as cryo - electron microscopy ( cryo - em ) reconstructionat 15  resolution demonstrating a similar structure for human pcc . 
 the structure defines the overall architecture of pcc and reveals unexpectedly that the  subunits are arranged as monomers in the holoenzyme , decorating a central 6 hexamer . 
 a hitherto unrecognized domain in the  subunit , formed by residues between the bc and bccp domains , is crucial for interactions with the  subunit . 
 we have named it the bt domain . 
 the structure reveals for the first time the relative positions of the bc and ct active sites in the holoenzyme . 
 they are separated by approximately 55  , indicating that the entire bccp domain must translocate during catalysis . 
 the bccp domain is located in the active site of the  subunit in the current structure , providing insight for its involvement in the ct reaction . 
 the structural information establishes a molecular basis for understanding the large collection of disease - causing mutations in pcc , and also has important relevance for the holoenzymes of other biotin - dependent carboxylases , including 3-methylcrotonyl - coa carboxylase ( mcc ) 57 and eukaryotic acetyl - coa carboxylase ( acc ) 8,9 . 
Methods summary
Crystallography
Cryo-electron microscopy
Mutagenesis and kinetic studies
Supplementary Material