SHELXE command line parameters

Experimental phasing with SHELXE

A typical SHELXE job for SAD, MAD, SIR or SIRAS phasing could be:

shelxe xx xx_fa -s0.5 -z -a3

where xx.hkl contains native data and xx_fa.hkl, which should have been created by SHELXC or XPREP, contains FA, σ(FA) and α. The heavy atoms are read from xx_fa.res, which can be generated by SHELXD or ANODE. 'xx' and 'xx_fa' may be replaced by any strings that make legal file names.

Normally the heavy atom enantiomorph is not known, so SHELXE should also be run with the -i switch to invert the heavy atoms and if necessary the space group; this writes files xx_i.phs instead of xx.phs etc., so the two hands may be run in parallel.

-a sets the number of global autotracing cycles.

-n imposes NCS during tracing, e.g. -n6 for six-fold NCS or -n if the number of copies is not known. The NCS operators are derived from the heavy atom positions or from the .pda file (see below). -n may now take the form: -n102, -n103, -n104 etc. for N-fold NCS with ONE heavy atom in each monomer, -n202, -n203, -n204 etc. for N-fold NCS with TWO heavy atoms in each monomer etc. This requires proper NCS. -n2, -n3 -n4 etc. specify the number of heavy atoms per monomer with either proper or improper NCS.

NCS may now also be specified in the .pda file to trace from a partial structure:
should be inserted before each monomer. The monomer is terminated by the next such GROUP BEGIN record or by:
Each monomer should then contain the same atom names in the same order, but the chain IDs and residue numbers may differ. The NCS operators are deduced from the coordinates of the monomers in the .pda file and are applied during tracing. The -n command line switch is not required.

If the heavy atoms are present in the native structure (e.g. for sulfur-SAD but not SIRAS for an iodide soak) -h is required (or e.g. -h8 to use only the first 8).

-z optimizes the substructure at the start of the phasing, starting from the sites selected by -h (if present, otherwise it starts from all sites in the .res file). -z can often make the difference between success and failure!

If -x is specified and a PDB-format reference file name.ent is provided, the origin shift and mean phase errors are calculated 'on the fly'. For experimental phasing the origin shifts are also used to identify which atoms in the reference .ent file are closest to the heavy atom sites, taking symmetry into account. This is a quick way of checking whether the substructure is correct, but note that it may be necessary to invert it with -i

-IN does N cycles density modification in global cycle 1 using free lunch reflections if set by -e. Subsequent cycles use the -m setting as before. In cases suitable for a free lunch one could try e.g. -I200 or more.

Expanding and verifying a MR solution with SHELXE

To start from a MR model without other phase information, the PDB file from MR should be renamed (to xx.pda to match the xx.hkl native data file) and input to SHELXE, e.g.

shelxe xx.pda -s0.5 -a20

The number of tracing cycles is usually more than for experimental phasing to reduce model bias. If the MR model is large but does not fit well, -o should be included to prune it before density modification by eliminating individual residues to optimize the CC for the model against the native data. For large structures, this optimization may be restricted to groups of two or more residues to fit into the available computer memory; the -u switch may be used to fine-tune the memory allocation for this.

-KN keeps the starting fragment for the first N global cycles, after that it is thrown away. If -K is given without a number or that number is larger than specified with -a, the initial (pruned) fragment is also still output to the .pdb file. The fragment does not have to be a polypeptide.

Tracing from an MR model requires a favorable combination of model quality, solvent content and data resolution. However, if the CC against the native data exceeds 25% after several cycles of autotracing, it is almost certain that the structure is solved! It is necessary to do several cycles to reduce model bias, the CC after the first cycle may be artificially high.

MRSAD - combining MR and experimental phasing

If both a MR model and anomalous data are available, but neither are good enough to give a good model on their own, the two approaches may be combined. To solve a structure in this way with MRSAD, first SHELXC must be used to generate the file xx_fa.hkl as for experimental phasing, then:

shelxe xx.pda xx_fa -s0.5 -a10 -h -z

This uses phases from the MR model to generate the heavy atom substructure, which can be optimized with -z as with experimental phasing. The substructure is then used to derive SAD phases that are combined with the phases from the MR model. The -o, -z and -h flags are often needed for this mode. This approach ensures the the heavy-atom substructure refers to the same choice of unit-cell origin as the MR fragment.

Using phases from other sources

If approximate phases are available, SHELXE may be used to refine them and make a poly-Ala trace:

shelxe xx.zzz -s0.5 -a3

where zzz is phi (phs file format), fcf (SHELXL LIST 6 format) or hlc (Hendrickson-Lattman coefficients, e.g. from SHARP or BP3). However it is recommended that if heavy atoms are found by SHELXD, they should be input directly into SHELXE with the -z switch to refine them before phasing, rather than using some other program to derive phases from the heavy atoms and input these phases to SHELXE. Reading in the heavy atoms has several advantages, for example they are used to make a 'no-go map' so that SHELXE does not trace the main-chain through them!

SHELXE output files

In all cases, native data are read from xx.hkl in SHELX format, and the final phases are output to xx.phs (or xx_i.phs if -i was set). The listing file is xx.lst (or xx_i.lst). If both xx.hkl and xx_fa.hkl are read, substructure phases are output to xx.pha (or xx_i.pha) and the revised substructure is written to xx.hat (or xx_i.hat). If -o is used to improve a model read in from xx.pda, the revised model is output to xx.pdo.

Alphabetical list of SHELXE options (defaults in brackets)

-aN - N cycles autotracing [off]
-AX - maximum random initial rotation in deg. for -O [-A3.0]
-bX - B-value to weight anomalous map (xx.pha and xx.hat) [-b5.0]
-cX - fraction of pixels in crossover region [-c0.4]
-dX - truncate reflection data to X Ångstroms [off]
-eX - add missing 'free lunch' data up to X Ångstroms [dmin+0.2]
-f - read F rather than intensity from native .hkl file [off]
-FX - weight for adding fragment phases each cycle [-F0.0]
-gX - solvent gamma flipping factor [-g1.1]
-GX - FOM threshold for accepting new peptide when tracing [-G0.5]
-h or -hN - (N) heavy atoms also present in native structure [-h0]
-i - invert space group and input (sub)structure or phases [off]
-kX - minimum height/sigma for heavy atom sites in xx.hat [-k4.5]
-KN - keep starting fragment unchanged for N global cycles [off]
-K - keep fragment unchanged throughout
-lN - reserve space for 1000000N reflections [-l2]
-mN - N iterations of density modification per global cycle [-m20]
-n or -nN - apply N-fold NCS to traces [off]
-o or -oN - prune up to N residues to optimize CC for xx.pda [off]
-q - search for alpha-helices as well as tripeptides [off]
-rX - FFT grid set to X times maximum indices [-r3.0]
-sX - solvent fraction [-s0.45]
-tX - time factor for helix and peptide search [-t1.0]
-uX - allocable memory in MB for fragment optimization [-u200]
-vX - density sharpening factor [default dependent on resolution]
-wX - add experimental phases with weight X each iteration [-w0.2]
-x - diagnostics, requires PDB file xx.ent with same origin [off]
-yX - highest resol. in Ångstroms for calc. phases from xx.pda [-y1.8]
-zN - substructure optimization for a maximum of N atoms [off]
-z - substructure optimization, program decides how many atoms [off]