Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

REFINEMENT PROTOCOL--rigid body refinement DETAILS--The domains were fit using Chimera. The model was refined using Phenix real-space refinement.

A total of 21100 particles were picked from ice images and CTF-corrected using Ctfit. Initial three-dimensional models were generated from either reference-free class averages or from a cylinder. The two models were essentially similar. Three noise-seeded models were generated and used as initial models in Multirefine. A model with two Ros-1 Fabs bound emerged from a population of 15000 particles and was subjected to further refinement using standard iterative projection matching, class averaging, and Fourier reconstruction. The final three-dimensional map with C2 symmetry was generated from 12373 particles.

20 mM sodium phosphate, 150 mM sodium chloride

Bovine rod PDE6 holoenzyme in complex with the Fab fragment of monoclonal antibody ROS-1

Rod cGMP-specific 3',5'-cyclic phosphodiesterase

Fab fragment of monoclonal antibody ROS-1

Objective lens astigmatism was corrected at 100,000 times magnification.

400 mesh glow-discharged Quantifoil grids with 2.0 Angstrom holes

Applied 3 uL of sample per grid and blotted for 1 second before plunging into liquid ethane (FEI VITROBOT MARK III).
Applied 3 ul of sample per grid, blotted for 1 second before plunging

phosphodiesterase 5/6 chimera catalytic domain

phosphodiesterase 6 gamma subunit inhibitory peptide

GafA domain of cone phosphodiesterase 6C

cGMP-specific 3',5'-cyclic phosphodiesterase, cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha' chimera

Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma, GMP-PDE gamma

cGMP-dependent 3',5'-cyclic phosphodiesterase

cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha'

DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLMIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQS
EDLADYFCQQYSSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSATDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

EVQLQQSGAEVVRSGASVKLSCTASGFNIKDYYIHWVKQRPEKGLEWIGWIDEIGDTEYVPKFQGKATMTADTSSNTAYL
QLSSLTSEDTAVYYCNAGHDYDRGRFPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTW
NSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD

GSHMEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNV
AYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCL
MILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITK
PWPIQQRIAELVATEFWEQGDLERTVLQQQPIPMMDRNKRDELPKLQVGFIDFVCTQLYEALTHVSEDCFPLLDGCRKNR
QKWQALAEQQ

QKLKCECQALLQVAKNLFTHLDDVSVLLQEIITEARNLSNAEICSVFLLDQNELVAKVFDGGVVDDESYEIRIPADQGIA
GHVATTGQILNIPDAYAHPLFYRGVDDSTGFRTRNILCFPIKNENQEVIGVAELVNKINGPWFSKFDEDLATAFSIYCGI
SIAHSLLYKKVNEAQYRSHLANEMM

MRLEECNILFELLTEIQDEAGSMEKIVHKTLQRLSQLLAADRCSMFICRSRNGIPEVATRLLNVTPTSKFEDNLVNPDKE
TVFPLDIGIAGWVAHTKKFFNIPDVKKNNHFSDYLDKKTGYTTVNMMAIPITQGKEVLAVVMALNKLNASEFSKEDEEVF
KKYLNFISLVLRLEHHHHHHHHHHHH

Cryo-electron microscopy reconstruction of bovine rod PDE6

THE IMAGED PDE6 WAS DERIVED FROM BOS TAURUS, BUT SOME MODELED SEQUENCES ARE FROM HOMO SAPIENS (CHAINS B, C, F, G) OR GALLUS GALLUS (CHAINS 1, 2). THE PHOSPHODIESTERASE 2A STRUCTURE WAS DETERMINED WITH FULL-LENGTH PROTEIN, BUT ONLY RESIDUES 387-571 WERE USED IN THE FITTED MODEL (CHAINS C, G).

IgG1-kappa 2E8 light chain, IgG1-kappa 2E8 heavy chain, phosphodiesterase 5/6 chimera catalytic domain, phosphodiesterase 6 gamma subunit inhibitory peptide, GafB domain of phosphodiesterase 2A, GafA domain of cone phosphodiesterase 6C

Phosphodiesterase, photoreceptor, PDE6, HYDROLASE-IMMUNE SYSTEM complex

MRLEECNILFELLTEIQDEAGSMEKIVHKTLQRLSQLLAADRCSMFICRSRNGIPEVATRLLNVTPTSKFEDNLVNPDKE
TVFPLDIGIAGWVAHTKKFFNIPDVKKNNHFSDYLDKKTGYTTVNMMAIPITQGKEVLAVVMALNKLNASEFSKEDEEVF
KKYLNFISLVLRLEHHHHHH