Protein production and assembly assays
Bovine clathrin heavy chain residues 1074–1675 (hub) were expressed and purified as described (Liu et al., 1995
). Bovine neuronal clathrin light chain b (LCb) used in crystallography was expressed and purified as described (Ybe et al., 1998
). Human neuronal LCb was expressed and purified as a his-tagged fusion protein for assembly assays, FRET assays and SPR. His-tagged LCb behaved identically to untagged bovine LCb (data not shown). Assembly assays were performed as described (Ybe et al., 1998
Crystal growth and data collection
Crystals were grown in 200mM citrate, 16–22% glycerol, 2% trifluoroethanol. Data was collected at the Advanced Light Source beamline 8.3.1. To collect low resolution data, two separate data sets were collected. The highest resolution reflections were collected using standard procedures. To gain adequate separation of the very low resolution reflections in the second data set, the detector was moved to the maximum distance from the crystal. With no further modifications the majority of the expected low resolution reflections could be accurately measured.
Data for the I4122 spacegroup was originally processed to 8.3 Å with an I/sigma I of approximately 4 using DENZO. To improve the resolution, maps were calculated using phases calculated from a molecular replacement solution (described below). Data was then processed to higher resolution in 0.1 Å or 0.2 Å steps, using HKL2000, despite worsening processing statistics and maps recalculated. This iterative process continued until visual inspection of the maps showed no improvement.
Molecular replacement model development
To generate an accurate model for molecular replacement, comparative models of the clathrin heavy chain repeats were generated. Structural alignment to the cryoEM structure (pdb code 1×14), loop modeling and energy minimization were used to build a complete model of CHC. This all atom model was truncated to a trimer of residues 1280–1630 and used successfully to find a molecular replacement solution where more simplistic search models had failed.
Phasing and Refinement of Crystal Data
The following strict guidelines for building into maps were followed to help prevent over-interpretation of the low resolution information. All new structural elements were built into 2Fo-Fc map density scaled to 0.06 e/Å3 (1.2 σ) or higher with positive Fo-Fc density associated with it. Novel regions from CLC were built as poly-alanine helices due to the low resolution of the maps. Since the CLC was not a continuous chain, the numbering of newly built residues is arbitrary. Building and numbering of clathrin heavy chain was based upon the refined all atom model used for molecular replacement.
To improve the maps prior to model building for the 7.9 Å (I4122) data, two steps were taken. First one round of rigid body refinement was performed using REFMAC, with each leg within the molecular replacement search model defining a rigid body. This was performed to be sure the position of each leg was correct within the trimer. NCS averaging of maps (FOM weighted phases with observed amplitudes) generated from the first round of rigid body refinement, across all three legs, caused density near the N-terminus of CHC to disappear, while density for the helical region of clathrin light chain became more defined (Figure S2B). NCS averaging all possible combinations of two legs led to determination that one randomly assigned pair of legs retained density at the N-terminus (Figure S2B). This suggests that these two legs are very similar in conformation while the third (chain C) is different.
Adjustments to the model were initially followed by simple rigid body refinements with two domains for each leg and the domain boundary at the flexible knee joint. Further rigid body refinements after model adjustments occured with increasingly smaller domains strongly restrained by the NCS. Details of the domain boundaries and NCS restraint parameters can be found in the supplementary information.
Subsequent refinement consisted of NCS restrained rigid body refinement alternating with NCS restrained domain B factor refinement. Final rounds of refinement, used to help minimize model bias, were achieved through simulated annealing with individual helices as rigid body groups while maintaining NCS restraints. The NCS restraints were gradually decreased through the last rounds of simulated annealing. Overall less than 2000 parameters were refined against greater than 10,000 reflections. Maps calculations for refinement steps were either with CNS 1.2 including default optimization of bulk solvent mask scaling parameters or with REFMAC with solvent scaling parameters, ionic radius and shrink equal to 2.0. This was necessary for calculation of high quality maps similar to previously determined low resolution structures (DeLaBarre and Brunger, 2003
). The 9 Å (P42
2) data was phased by molecular replacement and minimally refined with standard rigid body refinements.
Testing for model bias with decoy building
The intentional addition of helices outside but near 2Fo-Fc map density for the 7.9 Å (I4122) was used as a test for building with minimal model bias (Figure S2D). Helices placed in density weaker than 0.9 σ consistently appeared in negative density upon refinement and produced higher Rwork or Rfree values. The density, especially that of the N-termini of the clathrin light chains, represents the position of the domain relative to other domains of both CLC and CHC.
Modeling complete triskelia by combination of new crystallographic data with cryoEM data
Structural alignments between elements from the crystal structure reported here and the cryoEM structure by Fotin et al. (pdb 1XI4) were used to model full triskelia. Full legs were built by aligning the distal leg (residues 1–1130 of the cryo-EM lattice) with residues 1074–1130 from the crystal structure, which are N-terminal to the hinge point in the knee region (red arrowheads, ). Three straight legs or three bent legs were trimerized by alignment with the C-terminal part of the hub. Crystal structures and data have been deposited to the Protein Databank with codes 3LVG and 3LVH.
Förster resonance energy transfer
FRET assays were performed by labeling a double mutant of LCb (S9C and C190S) with a mixture of Alexa 555 and Alexa 647 fluorescent dyes and measuring steady state energy transfer under assembly or disassembly conditions.
Surface plasmon resonance
For SPR analysis LCb was covalently coupled to the surface of a CM5 flow cell (Biacore) and clathrin hub was flowed over the surface. Data was collected on a Biacore T100 biosensor and processed with Scrubber (University of Utah, Center for Biomolecular Interactions).