Tetrairidium, a four-atom cluster, is readily visible as a density label in three-dimensional cryo-EM maps of proteins at 10-25 A resolution.
Heavy metal clusters derivatized to bind to designated chemical groups on proteins have great potential as density labels for cryo-electron microscopy. Smaller clusters offer higher resolution and penetrate more easily into sterically restricted sites, but are more difficult to detect. In this context, we have explored the potential of tetrairidium (Ir(4)) as a density label by attaching it via maleimide linkage to the C-terminus of the hepatitis B virus (HBV) capsid protein. Although the clusters are not visible in unprocessed cryo-electron micrographs, they are distinctly visible in three-dimensional density maps calculated from them, even at only partial occupancy. The Ir(4) label was clearly visualized in our maps at 11-14 A resolution of both size variants of the HBV capsid, thus confirming our previous localization of this site with undecagold (Zlotnick, A., Cheng, N., Stahl, S. J., Conway, J. F., Steven, A. C., and Wingfield, P. T., Proc. Natl. Acad. Sci. USA 94, 9556-9561, 1997). Ir(4) penetrated to the interior of intact capsids to label this site on their inner surface, unlike undecagold for which labelling was achieved only with dissociated dimers that were then reassembled into capsids. The Ir(4) cluster remained visible as the resolution of the maps was lowered progressively to approximately 25 A.
Cheng
N
N
Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, USA.
Conway
J F
JF
Watts
N R
NR
Hainfeld
J F
JF
Joshi
V
V
Powell
R D
RD
Stahl
S J
SJ
Wingfield
P E
PE
Steven
A C
AC
D002213:Capsid / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000379:methods*; D006515:Hepatitis B virus / Q000737:chemistry / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007495:Iridium / Q000737:chemistry*; D015335:Molecular Probes; D050607:Organogold Compounds; D009942:Organometallic Compounds / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000648:ultrastructure*
D015335:Molecular Probes; D050607:Organogold Compounds; D009942:Organometallic Compounds; D014764:Viral Proteins; D007495:Iridium; C035797:undecagold