C. thermophilum Pyruvate Dehydrogenase Complex Core

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.

The initial model was used to generate our model using MODELLER. This inital model was fitted into the density using ChimeraX and refined in real space using PHENIX.

Native 60-mer core of Pyruvate Dehydrogenase Complex

Chaetomium thermophilum var. thermophilum DSM 1495

Automated selection with template derived from manual selection

Particle extraction, 2D/3D classification, Model refinement

For plunging, blot force 2 and blotting time of 6 sec were applied.

Acetyltransferase component of pyruvate dehydrogenase complex

MLAQVLRRQALQHVRLARAAAPSLTRWYASYPPHTIVKMPALSPTMTSGNIGAWQKKPGDAITPGEVLVEIETDKAQMDF
EFQEEGVLAKILKETGEKDVAVGSPIAVLVEEGTDINAFQNFTLEDAGGDAAAPAAPAKEELAKAETAPTPASTSAPEPE
ETTSTGKLEPALDREPNVSFAAKKLAHELDVPLKALKGTGPGGKITEEDVKKAASAPAAAAAAPGAAYQDIPISNMRKTI
ATRLKESVSENPHFFVTSELSVSKLLKLRQALNSSAEGRYKLSVNDFLIKAIAVACKRVPAVNSSWRDGVIRQFDTVDVS
VAVATPTGLITPIVKGVEAKGLETISATVKELAKKARDGKLKPEDYQGGTISISNMGMNPAVERFTAIINPPQAAILAVG
TTKKVAVPVENEDGTTGVEWDDQIVVTASFDHKVVDGAVGAEWMRELKKVVENPLELLL

We isolated the protein complex by using only the heavy fractions of a Phenomenex Biosep SEC-s4000 with an upper exlcusion limit in the MDa-range.

We performed Cryo-TEM on a vitrified, fractionated cell extract, where, after systematic image analysis we ended up with an Icosahedral core for the Pyruvate Dehydrogenase Complex.
scanning transmission electron microscopy

It is known that the E2 protein of the Pyruvate Dehydrogenase Complex forms an icosahedral assembly, e.g. in the human counterpart (PDB-ID: 6ct0)

Dihydrolipoyl, transacetylase, E2, Pyruvate, TRANSFERASE