Model building and refinement

Not all peptide stretches were built in sequence in the initial model. Therefore, unknown residues were removed and a refinement in BUSTER-TNT resulted in a model giving Rcryst/Rfree of 24.78/32.36. Various chains in the third refined model (buccaneer03) were put into the right place manually using LSQKAB and Coot, which resulted in a trimer containing three chains (Figure 45). Chain A contained 409 residues, chain B 384 and chain C 392 out of 574 residues, indicating a 68% complete BetP trimer.

A sequence alignment (Clustal W 1.83) of chain A, B and C to the full BetP sequence showed the missing and incorrectly built parts (red letters in the original BetP sequence) and green stars indicate the sequence fit of chains A, B and C to the original BetP sequence:

BetP MTTSDPNPKPIVEDAQPEQITATEELAGLLENPTNLEGKLADAEEEIILEGEDTQASLNW A --- B --- C --- BetP SVIVPALVIVLATVVWGIGFKDSFTNFASSALSAVVDNLGWAFILFGTVFVFFIVVIAAS A ----PALVIVLATVVWGIGFKDSFTNFASSALSAVVDNLGWAFILFGTVFVFFIVVIAAS B ---IVLATVVWGIGFKDSFTNFASSALSAVVDNLGWAFILFGTVFVFFIVVIAAS C ---IVLATVVWGIGFKDSFTNFASSALSAVVDNLGWAFILFGTVFVFFIVVIAAS ****************************************************

BetP KFGTIRLGRIDEAPEFRTVSWISMMFAAGMGIGLMFYGTTEPLTFYRNGVPGHDEHNVGV A KFGTIRLGRIDET---VSWISMMFAAGMGIGLMFYGTTEPLTFYRNGVPGHDEHNVGV B KFGTIRLGRIDEA---SWISMMFAAGMGIGLMFYGTTEPLTFYRNGVPGHDEHNVGV C KFGTIR---SWISMMFAAGMGIGLMFYGTTEPLTFYRNGVPGHDEHNV-- ****** ***************************************

BetP AMSTTMFHWTLHPWAIYAIVGLAIAYSTFRVGRKQLLSSAFVPLIGEKGAEGWLGKLIDI A AMSTTMFHWTLHPWAIYAIVGLAIAYSTFRVGRKQLLSSAFVPLIGEKGAEGWLGKLIDI B AMSTTMFHWTLHPWAIYAIVGLAIAYSTFRVGRKQLLSSAFVPLIGEKGAEGWLGKLIDI C AMSTTMFHWTLHPWAIYAIVGLAIAYSTFRVGRKQLLSSAFVPLIGEKGAEGWLGKLIDI ************************************************************

BetP LAIIATVFGTACSLGLGALQIGAGLSAANIIEDPSDWTIVGIVSVLTLAFIFSAISGVGK A LAIIATVFGTACSLGLGALQIGAGLSAA--- B LAIIATVFGTACSLGLGALQIGAGLSAAN--- C LAIIATVFGTACSLGLGALQIGAGLSAA--- ****************************

BetP GIQYLSNANMVLAALLAIFVFVVGPTVSILNLLPGSIGNYLSNFFQMAGRTAMSADGTAG A ---NIGTAG B ---ISADGTAG C ---ADGTAG ****

BetP EWLGSWTIFYWAWWISWSPFVGMFLARISRGRSIREFILGVLLVPAGVSTVWFSIFGGTA A EWLGSWTIFYWAWWISWSPFVGMFLARISRGRSIREFILGVLLVPAGVSTVWFSIFGGTA B EWLGSWTIFYWAWWISWSPFVGMFLARISRGRSIREFILGVLLVPAGVSTVWFSIFGGTA C EWLGSWTIFYWAWWISWSPFVGMFLARISRGRSIREFILGVLLVPAGVSTVWFSIFGGTA ************************************************************

BetP IVFEQNGESIWGDGAAEEQLFGLLHALPGGQIMGIIAMILLGTFFITSADSASTVMGTMS A IVFEQNGESIWGDGAAEEQLFGLLHALPGGQIMGIIAMILLGTFFITSADSASTVMGTMS B IVFEQNGESIWGDGAAEEQLFGLLHALPGGQIMGIIAMILLGTFFITSADSASTVMGTMS C IVFEQNGESIWGDGAAEEQLFGLLHALPGGQIMGIIAMILLGTFFITSADSASTVMGTMS ************************************************************

BetP QHGQLEANKWVTAAWGVATAAIGLTLLLSGGDNALSNLQNVTIVAATPFLFVVIGLMFAL A QHGQLEANKWVTAAWGVATAAIGLTLLLSGGDNALSNLQNVTIVAATPFLFVVIGLMFAL B QHGQLEANKWVTAAWGVATAAIGLTLLLSGGDNALSNLQNVTIVAATPFLFVVIGLMFAL C QHGQLEANKWVTAAWGVATAAIGLTLLLSGGDNALSNLQNVTIVAATPFLFVVIGLMFAL ************************************************************

BetP VKDLSNDVIYLEYREQQRFNARLARERRVHNEHRKRELAAKRRRERKASGAGKRR A VKDLSNDVIYLEYREQQRFNARLA--- B VKDLSNDVI--- C VKDLSNDVIYLEY--- *********

Figure 45| Trimer chains A, B and C after ten building cycles. Top view on the trimer with chain A (blue) containing 409 residues, chain B (red) containing 384 residues and chain C (green) containing 392 residues. Unknown residues within the trimer and symmetry related fragments were deleted.

This model was good starting point for further manual model building. Errors, such as mis-built residues or frame shifts in sequence had to be identified and corrected, and missing helices and loops had to be completed; this was an iterative process of refinement, quality control and rebuilding. Together with imposed stereo-chemical rules (ideal bond length and angles), refinement is an automatic adjustment of model parameters, the atomic coordinates, to improve their fit to the experimental data.

After building most of the missing parts with COOT and O, the removal of several frame shifts resulted in an improved model (BetP_TRIMER_ABC.pdb).

Positive and negative difference densities from the (Fobs- Fcalc)-map were contoured at 3+ and used as guidance for further manual building.

Several runs were performed using difference amplitudes and their standard deviations, with and without NCS restraints to find the correct refinement strategy.

As a result, refinement was performed against the scaled amplitudes and their standard deviations derived from SHARP (FP and SIGFP) with strict NCS restraints, except for the C-terminal domain (see 10.8). Refinement of the model (BetP_TRIMER_ABC.pdb) under these conditions using BUSTER-TNT resulted in Rcryst/Rfree of 26/30. In both programs experimental phase restraints and strict NCS

restraints were applied throughout the refinement. Phenix.refine included three translation-liberation-screw (TLS) groups per chain (for TLS see section 3.3.12 and for TLS group definition see 10.7) and used convential NCS restrain, whereas BUSTER-TNT used Local Structure Similarity Restraints (LSSR) as NCS restrain and ideal helix restraints. Around 90 cycles of building and refinement, using phenix.refine and BUSTER-TNT, were needed to achieve the final model of BetP.

In the first rounds of refinement used BUSTER-TNT and REFMAC-5 were used in parallel. Refinement in REFMAC-5 ended in a local minimum, indicated by an increase of Rcryst/Rfree values, no improvement in electron density map and model geometry. This program was therefore not further used. After this decision, refinement was continued using phenix.refine and BUSTER-TNT alternately. All main refinement strategies and steps with their results are summerised in Table 25.

The combination of different refinement programs proved to be very useful:

phenix.refine improved the stereochemistry of the model and allowed further correction using the phenix.geometry_minimization tool by solely optimising stereochemistry properties of the model without including any observed data (electron density); and BUSTER-TNT resulted in better guiding (Fobs- Fcalc) electron densities, which allowed better judgment for model building. However, the qualities of the #A-weighted (mFobs- DFcalc) (2mFobs- DFcalc) electron density maps were of similar quality with both programs.

Table 25| Refinement and model building stages for SeMet-BetA.

STAGE OF MODEL MODEL BUILDING, STRATEGY &

PROGRESS PROGRAM

& STRATEGY TLS

groups^a) Rcryst Rfree RAMACHANDRAN^b)

buccaneer02 auto BUSTER-TNT - 25.14 36.48 -

-loop 2, 5 and 6 remodelled in O using lego-C!

- several outliers removed

Several rounds of manual building, correction of helices and loop regions, alternating refinement between phenix.refine and phenix.geometry_minimization with one round of simulated annealing, TLS refinement and BUSTER-TNT using LSSR NCS restraints.

64_refine manual BUSTER-TNT

-LSSR NCS, MLhl -target phenix.refine11

- 25.68 26.49 93% / 0.26%

a) TLS groups defined in 10.7 b) in most favoured regions/outliers as defined by MOLPROBITY

In the early stages of refinement, positive (Fobs- Fcalc) electron densities appeared in all three monomers. Aromatic residues around the density were superposed on the substrate-binding pocket of the glycine-betaine binding protein ProX from E. coli (pdb ID 1r9l) (Schiefner et al., 2004a), confirming the binding pocket architecture and glycine-betaine position in BetP.

Figure 46|Positive Fo-Fc electron density for glycine-betaine and the strategy for its positioning in BetP. a, side view on transmembrane helix 8 in chain A with 2Fo-Fc electron density map (blue) at 1.3! and the positive Fo-Fc density (green) countered at 2.4!, indicating the glycine-betaine position; b, corresponding top view on transmembrane helix 8 and 4; c, overlay of the glycine-betaine binding pocket of BetP (grey) and the periplasmic glycine-betaine binding protein ProX from E. coli (yellow). d, The sequence homology between BetP and ProX in the 4-helix bundle TM3, TM4/TM8, TM9 of BetP is shown on the right hand side.

The density of the substrate did not improve dramatically during the whole refinement and was different in the three monomers. B-factor analysis (Figure 48) in

the final model suggests different substrate occupancies within the trimer. However, occupancy refinement was not feasible with this electron density map quality.

Table 26| Data collection and final refinement statistics.

SeMet-BetA