If you are confused, use MORE 3 to inflate the lst-file.
With MORE 3, the resulting file
will contain a list of every single parameter, so that you can check.
Q26:
What is the number below the atom name after running a least-squares
matrix inversion ?
The number under the atom name is the 'radial atomic positional
esd'. It characterizes the error in the coordinates of an atom taking
into account the variance of the coordinates along the crystallographic
axes and the covariances (i.e. the off-diagonal elements of the
inverse of the Least-Squares matrix) between them. The exact formula
for how this is done is only documented in the source code.
If you want to calculate an error along a specific direction you
should divide this number by sqrt(3), as in such a case the errors
perpendicular to that direction do not matter.
Q27:
Some Waters are moving away from the centre of density toward the
corner of the density. What is going on ?
George: We have sometimes seen atoms move to the edge of density in a high
resolution refinement. We attribute this to (a) Fourier series
termination errors because of missing reflections (including Rfree
reflections), and (b) the weights for refinement are of necessity
different to those used to make a Fourier map (which theoretically
requires unit weights). In either case these refined positions are the
'correct' ones, the map is misleading. However anti-bumping (BUMP) and
other geometric restraints can also move atoms out of density, in this
case you need to check whether you need to include partial occupancies.
BUMP is not applied if the occupancy sum is less than 1.1.
Q28:
My substrate is pushed away by anti-bumping restraints that do not make
sense to me. What is happening ?
Make sure that you have chosen the correct scattering factor number
for all atoms in the substrate.
Short distances may be for real if the substrate is not fully occupied.
You can model this situation using multiple conformation (see other
questions in the FAQ).
Q29:
Our data/parameter ratio is about five, can we get rid of some restraints ?
George:
We always retain restraints for the protein. For those parts of the
structure that are well determined from the data, the data will carry a
much higher weight than the restraints. Leaving them in handles the less
well determined parts of the structure (e.g. Glu sidechains with high
B-values and disordered regions in general) without needing to take
special individual action for each of them. We usually leave the
restraints off for metal ions but not for organic substrates; the latter
may have high B-values or partial occupancies and so be in need of
restraints. If the structure of the substrate is in the CSD (or can be
determined by doing a small-molecule structure), you can use SHELXPRO to
generate the restraints automatically.
Q30:
What should be the final R-values of a protein structure at atomic
resolution
Depends on the problem ... Given good data (high completeness, reasonable
redundancy, I/sig(I) approx 2 in outer shell (with sigmas estimated
correctly)), Rwork should be below 12% with R
freenot much higher,
i.e. delta(Rwork,Rfree) < 3.0 percent.
There are a few structures with
Rfree lower than 10% and many with Rfree larger than 15%.
Q31:
Is it possible to refine a structure on two or more data sets measured
at two or more wavelengths?
Yes, using the LAUE and HKLF 2 instructions it is possible to have a
different wavelength for each reflection.
Q32:
SHELXL-93 gave esd's for bond angles involving riding hydrogen atoms,
SHELXL-97 doesn't - is this a bug or a feature ?
George:
The program SHELXL-93 calculated esds involving riding H-atom in a way
that was mathematically correct, assuming that the magnitude and
direction (but not position) of the X-H vector were exactly fixed; the
resulting esds thus depended on the combined scattering power of H and X
rather than just X. This gave rather small H-atom esds that confused
people, so I made the 97 version output zero esds for riding hydrogens. If
you want realistic esds for hydrogens you have to refine them freely (not
recommended for proteins)!
Q33:
How do I include the anomalous signal of Se-atoms into my refinement ?
Normally, macromolecules are refined with MERG 4, i.e. all
symmetry equivalents and Friedel-pairs are merged and all f" values are
set to 0.0. If there is a strong anomalous scatterer, like Se in a
seleno-methionine substituted protein, it may be better to keep the
Friedel mates apart and explicitly take the anomalous signal into account.
To properly treat the anomalous signal you have to provide f' and f" values
to SHELXL by using the DISP command.
It may be worth playing with different f' and f" values to get the
best results. I am interested in this myself; so if you make any
interesting observations in this respect, please let me know:
trs@shelx.uni-ac.gwdg.de
When you refine against anomalous data and you are using Rfree,
make sure that for all Friedel pairs either none or both reflections are
in the test set (this is a case similar to NCS, where correlated reflections
should be all in the test set to avoid artificially low values for
Rfree).
Q34:
I have been warned that SHELXL was not really written to refine
at 2.4 A resolution. Can I do it anyway ... ?
Maybe ... you will need to decrease the sigma on some restraints to keep
the model in order. For example, for SIMU restraints the default of
0.1 is probably to loose and should be set to something between, let's say
0.05 and 0.02. This will probably lead to an increase in the R-value,
but may be benefical to your free R-value (and you model :-)).
Q35:
After a few small changes, my refinement jobs against 0.83 A data all
over sudden always blow up with an error message ** REFINEMENT UNSTABLE **. How can I fix this ?
In many cases, unstable refinements are difficult to fix.
Sometimes removing the atoms giving rise to the maximum
shifts will improve things. If this does not work, I normally
use a STIR instruction of, let's say, STIR 1.1 0.05. This
has helped in most cases.
Q36:
SHELXL complains that that I have too many atoms on a single SIMU/DELU
instructions. What can I do ?
George:
This limit is more or less impossible to increase, but you can simply
split the offending DELU (and possibly SIMU) instructions, e.g.
DELU N_1001> OT2_9999 ! One chain per DELU, identify by first and last atoms
DELU O_1 > O_999 ! Waters, cations etc. - can split further if required
When I originally wrote the program for small molecules, I thought that
this limit would be fairly safe, but it never pays to hardwire such
limits in, however much easier it makes writing the code.
Q37:
I have to do a refinement of a protein containing some unusual cofactors and
ligands. What is the best approach to create a parameter file ?
You can use the 'J' option in SHELXPRO to create SHELXL restraints for
any fragment for which you have coordinates in a file in PDB, CSD or
SHELXL ins-format.
If you want, I'd be happy to put your parameter set on our web site.
Q38:
How do I restrain an SCN molecule to be straight ?
It is difficult to restrain a linear molecule to be straight.
A very small sigma on the DFIX and DANG restraints envolved will make
the molecule almost straight. But as the restraint is not very sensitive
to small deviations from linearity, there
will always be a small distortions. Making the sigma extremely small
(i.e. < 0.001 or so) will not have any effect, as SHELXL has an
internal cut-off for small sigmas to avoid problems during minimization.
The best solution probably is to put SCN into the refinement as
a rigid group with ideal geometry (from CSD or other source) and
only refine the translational and rotational parameters of that
group (AFIX 6, manual page 7-15). Refinement with AFIX 9 (page
7-15) would allow the bondlengths to shrink or expand
uniformly - this would give the model a chance to take librational
effects etc into account (not really, but better than nothing ...).
Q39:
Any suggestions for restraints for glycerol ?
Here is what I (TRS) use:
DFIX_ALK 1.417 CA OA CC OC ! Engh&Huber CH2E-OH1
DFIX_ALK 1.530 CA CB CB CC ! Engh&Huber CH1E-CH2E
DFIX_ALK 1.433 CB OB ! Engh&Huber CH1E OH1
DFIX_ALK 2.431 CB OA CB OC CA OB CC OB ! shelxl DFIX_SER CA OG
... no guarantees, as always ...
Q40:
How do I straighten out an azide molecule ?
George:
There is no easy way to restrain it effectively so I recommend making the
esds of the DFIX and DANG instructions for the azide very small (e.g.
0.001). For the record, there is a difficult way involving 9 extra free
variables and three SUMP restraints. FLAT doesn't help - all groups of three
atoms are coplanar - so it can be removed.
Q41:
How do I generate a restraint across a symmetry element ?
George:
To apply a restraint accross a symmetry element you will need to specify
an Eqiv instruction and then refer to the symmetry generated atom(s)
using _$1 etc. For example, if a disulfide bond invloving SG of Cys29
and its symmetry equivalent is bisected by a crystallographic twofold
axis at 0.5, y, 0.5 you need to specify:
EQIV $1 1-x, y, 1-z
DFIX_29 2.031 SG SG_$1
DANG_29 3.035 CB SG_$1 SG CB_$1
For the disorder you should specify PART numbers and you should use a
free variable for the occupancies (starting value on the FVAR
instruction, e.g. 21 and -21 instead of 11 for occupancy) so that you
can constrain the sum of occupancies to one. This is all fully explained
in the documentation!
Q42:
How do I generate restraints for a linear arrangement of atoms,
i.e. a cyano-group ?
There is no standard way to generate such restraints in SHELXL.
Here is the recommendation from George:
To make a cyano group into a rigid group, put it immediately after the
atom to which it is attached, put an AFIX 6 instruction before that atom
and AFIX 0 after the N. If there are hydrogens attached to the first
atom, the AFIX instructions should be combined using 5 as the code for a
subsequent atom in a rigid group, e.g.
AFIX 6
C12 ...
AFIX 25
H12A ...
H12B ...
AFIX 5
C ...
N ...
AFIX 0
The initial geometry of the C12-C-N group will be retained, so it is
important to start from an accurate geometry, obtained e.g. by a rigid
group fit of an accurately detemined small molecule structure (either
using FRAG...FEND etc. in SHELXL or by some other program).
The 'standard restraints' are unlikely to be adequate for most ligands,
they will often leave parts of it unconstrained and it will fly apart.
The recommended procedure is to search the Cambridge database for a
suitable small molecule structure (or if there isn't one, actually grow
crystals of the ligand and determine its structure - SHELX works for
small molecules too!) then use the J option in SHELXPRO to generate
restraints form the atom coordinates. You may still need to add some
FLAT and CHIV 0 restraints by hand, but usually it is obvious where.
Q43:
When should I put Hydrogen atoms ?
Don't put hydrogens before the very end - they do not improve
the phases much, but cost a lot of computer time. Do not put the hydrogens
you want to see (e.g. the ones on histidines). It does not count to first
put them, remove them and then proudly present Fo-Fc density for hydrogens.
There is a bias-problem, even for these small atoms.
Q44:
Can I put Hydrogens if I have good data to 1.6 A ?
George:
If you have good 1.6A data you can expect to 'see' some of the hydrogens.
In general these will be the N-H, CH and CH2 groups in which the N or
C atom has a low temperature factor. There is no harm in putting them
in if they reduce Rfree, because they do not add any extra parameters.
Q45:
Will putting Hydrogens introduce more parameters into my refinement ?
George:
Adding hydrogens using SHELX improves the model and does not add any
extra parameters, so the only reason not to do so is that is costs
computer time. For this reason we tend to add them late in the
refinement, after making the atoms anisotropic (if justified) and
modelling disorder. This has the advantage that the program usually adds
the right hydrogens automatically (using HFIX) even for disordered
residues.
However it is better NOT to add the hydrogens on -OH groups (Tyr, Ser
and Thr) because (a) it is rare that they can be seen in difference
maps, (b) they have little effect on the R-values etc., (c) it is
difficult for the program to predict their positions accurately, (d) if
the program accidentally assigns two hydrogens to the same H-bond and
then they are refined using the 'riding model' (AFIX 83), and
antibumping restraints are switched on (BUMP), the repulsion between the
hydrogens can introduce mechanical distortions into the structure.
Adding hydrogens to proteins usually reduces R1 and R1(free) by the same
amount, typically 0.5 to 1%. The effect can be bigger for accurate data
from small molecules, recently we had an example where adding the
hydrogens reduced R1 from 10.6% to 3.7%!
Q46:
How do I attach hydrogens to waters ?
George:If you can see them in a difference map then you
can insert the peaks and refine them with DFIX or SADI
restraints for O-H and H...H. If you can't
see them I doubt if it is worth trying to guess the positions,
someone might believe them.
Q47:
I have 0.95 A data and I can see hydrogens in 2Fo-Fc maps.
How do I make hydrogens anisotropic ?
You don't ... :-). Even for small small molecules, we NEVER refine
hydrogens anisotropically.
Q48:
When I add hydrogens, SHELXL shows a a warning
"** BAD AFIX CONNECTIVITY: N_1 BONDS TO CA_1 CD_1 **.
I did specify the N terminus when I ran shelxpro
in the first round of refinement and I checked that I had the
HFIX 33 N_1 statement before all other HFIX instructions.
What else should I do?
Your first residue is probably a proline, for which HFIX 33
does not make sense. To find the appropriate HFIX statements, try to
find something sensible in some other place in the ins-file and
use it for the Proline.
Q49:
The pdb file generated using the 'G' option (from .res) in
shelxpro doesn't have a distinction for e.g. the three gamma
hydrogens of Ile (they are all named 'HG2'). Bug or Feature ?
George: 'It is a problem with the PDB format, because only three characters may be
used for the name (an extra character is reserved for 2-letter element
names). Partly for this reason, we never deposit hydrogens, most programs
that need them recalculate their positions anyway.'
Q50:
After adding hydrogens, SHELXL complains:
"** BAD AFIX CONNECTIVITY: N_1 BONDS TO CA_1 **". What is happening ?
The program is trying to make the terminal N into an amide instead of
NH3+, which doesn't work because it only bonds to one atom.
Maybe you didn't specify the N terminus when you ran SHELXPRO? The
quick solution is to include: HFIX 33 N_1
before any other HFIX instructions. The program always applies the first
HFIX that is appropriate to a given atom.
Q51:
The hydrogens in my pdb-file have B-values of -94.75 A^2 - what is
going wrong here ?
When refined as 'riding hydrogens', the number in the B-factor field
of the .res-file is -1.2. If this value is not interpreted correctly
(i.e. the B-value of the hydrogen is constrained to be 1.2 times the
B-value of the parent non-hydrogen atoms), you get these strange
numbers (-1.2 times 8 x pi^2) = -94.75 . The best is to simply
ignore such B-values.
Q52:
When should I start modelling disordered atoms ?
- if you have atomic resolution data, don't do anything about disordered before
going anisotropic (why waste time interpreting suboptimal maps ?)
- if your data are not good enough for anisotropic refinement, first (in the ideal
world automatically) build most of your solvent, and then check the suspicious places.
Q53:
How can I find disordered parts of my protein ?
Look in the diagnostics tables produced by SHELXL for:
- difference peaks near residues (I usually check all peaks with abs(peak)>5.0
in a 1Fo-1Fc map systematically)
- negative electron densities at atomic sites
- high B-values
- SIMU/DELU restraint violations
Q54:
How do I get a disordered residue under control ?
to model a disordered sidechain:
- Group everything from CB onwards by using a PART instruction.
- Reset B's to isotropic.
- Set occupancies to 0.66 - the first conformation you find is usually the
major one. Therefore 0.66 is a good starting point.
- Refine for a few cycles starting with a few cycles of isotropic refinement then
anisotropic (use the second parameter of the CGLS statement for this).
If you are using the second parameter of CGLS for Rfree, forget about the
isotropic refinement or do it in a separate job if necessary.
- Try to pick up the second conformer in 1fo-1fc maps.
- Put in the second conformer and couple its occupancy to the first by using free
variables (FVAR). The 'u'-option in SHELXPRO is useful for this.
- If you use the correct nomenclature and PART instructions, SHELXL will put the
the necessary restraints automatically.
Q55:
What do I do with a sidechain for which the electron density is a complete
mess ?
We generally, try to model all disorders, but if we really can't see anything
we truncate the sidechain.
SHELXL usually doesn't mind this,
except that it may not be able to place hydrogen atoms, e.g. if you cut
at CB, the program cannot make an idealized CH2 out of it.
The remedy is either to change the name of the residue to ALA (very confusing !) or to
override HFIX with say HFIX_resnum 0 CB (or HFIX_resnum 3 CB),
which must come before the original HFIX. The latter is the better.
Q56:
The most important atom in my structure is the OG of a Serine. It is
not really in one position, but no matter what I do I cannot find any
density for a second conformer. I would like to locally release the
SIMU restraint to let it the OG loose a bit. But it does not work ...
This is a bit tricky. If there is two SIMU restraints addressing the same pair
of atoms, the program keeps whichever allows the smallest deviation.
There is a trick to get around this. Let's say, your Ser has residue number 5.
Then you should:
- outcomment the normal SIMU statement:
REM SIMU 0.1 $C_* $N_* $O_* $S_*
- generate the restraint for the serine CB-OG:
SIMU 0.3 0.6 1.7 CB_5 OG_5
- generate the other restraints without regerating the CB-OG
(this assumes that the order of atoms in the file is ... CB_5 OG_5 N_6 ...):
SIMU 0.1 N_1 > CB_5 N_6 > LAST
This will give the following local restraint:
0.6000 CB_5 - OG_5
The 0.6 is used here because OG is a terminal atom.
Q57:
I have a sidechain in two alternate positions. A water molecule
is consistent with position one, but will clash with position two.
What can I do ?
You need to couple the water and the tyrosine disorders
using PART numbers and use a free variable for the occupancy.
Q58:
I have a disulfide that looks like it might only be partially in the
reduced state. How can I model it ?
Thomas Pape in our lab had the same problem. He used PART 1 for the
unbroken part. PART 2 (for which restraints are automatically
generated) for the part that was broken, but still in disulphide
position and PART 3 for the part of the disulphide bridge that was
broken and had moved away. This way, atoms in PART 3 are not affected
by the disulphide restraints.
You may need to create some extra restraints for PART 3 but the rest
should work automatically.
Hope this is not too confusing ...
Q59:
I have a Lysine in my structure that sometimes forms a Schiff base and
sometimes not. How can I model this ?
This is one of the rare case where you need to apply different
restraints to different conformations of the same side chain
(normally, if you have to do this, something is fishy ...).
As there is currently no way to put restraints specifically for
PART identifiers, you will have to rename the two conformations
into artificial residues, let's say, ALYS and BLYS for which you
then can speficy all the restraints you need. Having ALYS for the
non-Schiff base Lysine will also allow you to use special non-standard
Lysine restraints for that particulare amino acid.
Q60:
I managed to partially photoactivate my crystal, can I somehow model
the ground state and the activatated state simultaneously ?
George:
In principle you can make the whole of the ground state PART 1 and the
whole of the excited state PART 2. The occupancies can be refined in the
usual way with free variables. Either PART can be fixed or refined
freely using AFIX instructions. It becomes appreciably more complicated
(but still possible) if you wish to model disordered side-chains as well
(you need to use extra PART numbers, plus some BIND instructions) or
when you include hydrogens (you need to modify the AFIX instructions
[that are generated from HFIX] by hand if you wish to keep parts of the
structure fixed).
Although this all works - we recently refined a 20 amino-acid peptide
for which the whole molecule was in two separate conformations on top of
one another - it is not trivial and could result in an awful mess.
Q61:
How can I interface with O ?
- run the following shellscript on your pdb-file: pdb2o.sh
- load both resulting files into O, color one in green, one in
red, ordered parts will then show up in yellow.
- put the occupancy datablock into your .message_template
in O: write .message_template tmp ;
then edit tmp to be:
.MESSAGE_TEMPLATE T 6 40
%MOLNAM %RESNAM %Restyp %ATMNAM, xyz =
atom_xyz
; B =
atom_b
; Occ =
atom_wt
then back to O:
read tmp
- when working with O, use the undocumented fileformat 'BUD' to
write fractional coordinates.
Q62:
How are alternative conformations marked in the pdb-files written by SHELXL ?
SHELXL-97 strictly obeys pdb-rules for alternative conformations,
i.e. a disordered serine would look like:
ATOM 749 N SER 98 -47.798 18.206 40.432 1.000 10.05
ATOM 750 CA SER 98 -47.369 18.674 39.124 1.000 10.02
ATOM 751 C SER 98 -46.129 17.884 38.664 1.000 8.53
ATOM 752 O SER 98 -45.304 17.445 39.458 1.000 10.25
ATOM 753 CB ASER 98 -46.887 20.145 39.241 0.456 10.19
ATOM 754 OG ASER 98 -47.931 20.977 39.669 0.456 10.51
ATOM 755 CB BSER 98 -46.911 20.164 39.247 0.544 10.53
ATOM 756 OG BSER 98 -46.430 20.638 37.992 0.544 12.85
the identifiers (" ",A,B,...) can be mapped 1-to-1 to the PART
instruction used in input files for refinement.
Q63:
How can I work with alternative conformations in O ?
the easiest way to deal with double-conformations is probably
to read in two molecules each corresponding to one conformation,
work on them as usual, save both of them, and then do some manual
cutting and pasting ...
Following is a shell-script to make two pdb-files out of one
(written for SHELXL-97 output, but easily changeable for other
formats).
#/bin/csh -f
#
# filename: pdb2pdbo.sh
#
# convert SHELXL96-pdb
# to something useful for O
#
# Thomas Schneider 2-Feb-96
#########################################
#
# some cleanup on pdb-file
# ------------------------
grep -v ANIS $1.pdb \
| grep -v "W HOH" \
| grep -v ^CRYST \
| awk '(substr($0,14,1)!="H")&&(substr($0,13,1)!="H") {print}' \
> tmp1.pdb
#
# extracting Cryst1 card
#
echo Extracting CRYST1 from pdb-file
head -10 $1.pdb | grep ^CRYST1 > t_cryst
cat t_cryst
#
# separating multiple conformations
# -------------------------------
nawk ' \
BEGIN {OFS=""}\
{s1=substr($0,16,1);s2=substr($0,17,1)} \
(s2=="A") {print substr($0,1,16)," ",substr($0,18,50)} \
(s2==" ") {print} \
' tmp1.pdb > tmp1_A.pdb
nawk ' \
BEGIN {OFS=""}\
{s1=substr($0,16,1);s2=substr($0,17,1)} \
(s2=="B") {print substr($0,1,16)," ",substr($0,18,50)} \
(s2==" ") {print} \
' tmp1.pdb | grep -vi WAT > tmp1_B.pdb
foreach v (A B)
nawk ' \
BEGIN {OFS=""} \
{print substr($0,1,21),"'${v}'",substr($0,23,45)} \
' tmp1_${v}.pdb > tmp2
cat t_cryst tmp2 > $1.pdb${v}
end
BTW: using the undocumented 'BUD' format when writing coordinates will
write fractionals :-) .
Q64:
Why can't SHELXL use 0/1, 1/0, 0-999/0-999 to flag reflections belonging
to the free/work set ?
A word from the author of the program: 'One line of the .hkl file contains
h,k,l,I,sigma(I) and (optionally) a 'batch number'. The remaining space
on the card (!) is reserved for the direction cosines. This format has
remained unchanged since the late 1960's (Microsoft and CCP4 please
note) and under no circumstances whatsoever will I agree to a change
that could introduce an incompatibility (SHELX users appreciate my
obstinacy in such matters). The batch number is normally little used
(it was originally designed for film data) except for non-merohedral
twins (HKLF 5 format), in which case it becomes the component number.
The only reasonable and compatible way that I could introduce an Rfree
flag was to make the 'batch number' negative for the reference set and
positive for the working set. Since the default batch number is 1, this
means that the Rfree flag is usually +1 or -1.
Q65:
Why does SHELXL not model my low resolution data, even if I use the
bulk solvent correction ?
Possible reasons are:
- The solvent model implemented in SHELXL is quite primitive and
might not be adequate.
- Your low resolution data may not be very good.
- If you collected the data on a synchrotron and did not attenuate
the beam during the low resolution pass, you might have rotated too
fast for the stepping motor to keep up. For the phi-motors used at
EMBL-Hamburg, for example, to stay
on the safe side, one should not rotate faster than 1 deg./10 seconds.
If you rotate faster, the motor misses steps and screws up the data.
- Trouble might also be caused by erase lamps in imaging-plate scanners
that are not functioning properly. The very strong reflections will not
be erased completely causing problems for the reflections ending up
in the same place on the plate during the next exposure.
- You did not remove overloaded reflections.
Q66:
I have excellent data to 1.4 A.
R dropped from 19.3 to 16.3 and Rfree from 21.6 to 21.0 on
going anisotropic, obs/par went from 4.8 to 2.1
Can I go/stay anisotropic ?
Whether or not your data allow anisotropic refinement depends not only
on the resolution but also on solvent content (which affects the data to
parameter ratio), data quality and other factors.
The only way to find out whether anisotropic refinement is justified
is to do an Rfree-test.
Generally, if the drop in Rfree is less than 1 % you should revert to
isotropic.
We recently had a dataset measured (weakly) to 1.2 A that gave a 0.5 % drop
in Rfree so we switched back to isotropic. On the other hand we have one
1.4 A dataset (but the crystals would have diffracted further) where Rfree
dropped 3.5 % on going anisotropic.
Q67:
I have data to 1.35 A for a protein containing a heme. Rwork drops by
3.2 and Rfree by only 1.4 % on going anisotropic (approx 77000 data /
33000 parameters). What shall I do ?
Try to just refine the Fe and S atoms and perhaps the heme
anisotropically. Since this is many fewer atoms than for full
anisotropic refinement, a drop of say 0.4 % or better in Rfree
would be acceptable. If Rfree drops by even less, then you should go
back to the fully isotropic model.
Q68:
I have 1.6 A data, Rfree drops by 0.2% on going anisotropic. Is this
significant ?
In general, on would not regard a drop in Rfree of 0.2% to be significant,
since Rfree, which is based on relatively few reflections, has quite a
large standard deviation.
Q69:
On going anisotropic Rfree converges after 3 cycles, Rwork only
settles after about 10 cycles. Should I worry ?
Rfree always "converges" faster than R1 in cases where Rfree doesn't
drop by the same amount as Rwork. R1 should be allowed
more or less converge, knowing well that the structure is
"overfitted" to have a clear-cut situation.
Q70:
Rfree doesn't go down all that much on going anisotropic, but the maps
are much better. Can't I continue with anisotropic refinement ?
Your maps look 'better' because they look more like your model -
that's what overfitting is about.
Q71:
I get loads of 'may be split' messages. What does this mean ?
- 'may be split' messages tell you that the largest eigenvalue of the ADP-matrix
is more than three times larger than the smallest. In other words: the
corresponding ellipsoid looks like a cigar.
- If you have a lot of 'may be split' messages, either your DELU and SIMU
restraints are too slack or anisotropic refinement is not justified,
anyway.
- They can indicate places in your structure
where modelling of multiple conformations may be advisable.
- it is normal for a well-refined structure to still give a few
'may be split' messages.
Q72:
Some of the Uij's in the .res file and in the .pdb file match, although they are
in different order, others are completely different. Is this a bug or a feature ?
It is a feature. The order in the .res file is: 11 22 33 23 13 12 (see SHELXL-manual).
The order in the .pdb file is: 11 22 33 12 13 23 (pdb convention). The differences in
the numbers arise from the Uij's in the .res file being relative to the crystal axes
and the values in the .pdb file being relative to orthogonal axes and multiplied by
10000.
Q73:
Upon PAVARTI analysis of Shelxl anisotropically refined protein structures,
I find the majority of atoms with large anisotropy ( axis ratio < 1:5 ) are
from residues with alternate conformations. Could this be a systematic
problem due to the refinement of both occupancy and anisotropic thermal
parameters together?
George:
I suspect that it is generally true that atoms in disordered residues
have higher equivalent Uiso values and are more anisotropic. This is
chemically eminently plausible, but might also be influenced by the
types of restraint we apply. I note that the author of REFMAC prefers to
use an ISOR rather than SIMU type of restraint in such cases, maybe you
should try this (and see how it affects Rfree), since it would tend to
make the atoms less anisotropic.
Q74:
Is it possible to read in a partial structure
factor (F/phi), to be combined with the F/phi calculated from the atomic
coordinates ?
George: SHELXL does not have a way of reading in partial structure factors,
and it would be very difficult to modify the code to do this.
The bulk solvent correction is a weak link in SHELXL and I am trying to
find a way of improving it (as of 3-Dec-2000).
Q75:
I get very high R-values especially at low resolution (with Fobs
systematically weaker than Fcalc). Upon refining the Extinction
coefficient in SHELXL, Rwork and Rfree decrease dramatically.
Is this Extinction for real ?
George:
I have never encountered extinction with a protein crystal and (for good
theoretical reasons) don't expect to. Even with small molecules,
freezing the crystal often eliminates extinction very effectively. Note
that since the solvent and extinction affect the data in a very similar
way, it is not possible to refine both, so SHELXL is designed to make
this logically impossible (I frequently have to prevent users from doing
things that don't make sense). There are three common causes of large
apparent extnction (in order of frequency):
(1) You are reading in F (obtained by e.g. converting from an XPLOR input
file) but have specified HKLF 4 (for F-squared) in the .ins file.
(2) 'Overloads' have not been removed from the data.
(3) Your detector (or the data processing software) is faulty.
Q76:
I am refining a structure against twinned data. Can a structure with a
twin-ratio of 0.5 be refined ? Are the F's calculated in the .fcf-file
'detwinned' ? How can I check the progress of the refinement ?
George:
The .fcf file is 'detwinned', so you can make maps etc. in the normal
way using SHELXPRO. Generally the quality of such maps is not as good
as that of maps from normal not-twinned crystals at the same resolution,
but using SHELXL it is entirely possible to complete the structure
determination even if the twin factor is exactly 0.5. Some other
programs use detwinning methods which are mathematically unstable when
the twin factor approaches 0.5. Details of the algorithms used are
given in the manual. Exactly the same procedures can be used to monitor
the refinement as for untwinned crystals, but care is needed with Rfree
because twinning introduces correlations if one of a twin-related pair
of reflections is in the working set and the other is in the reference
set. You can use the thin-shell method of choosing the reference
reflections to get around this (in the V option in SHELXPRO).
One more thing: refining against twinned data it can be very dangerous
to use automatic structure extension methods that are based on
difference Fouriers (i.e. ARP, SHELXWAT). When used with twinned data, such
methods tend to produce artifacts, especially in the early stages of a refinement and
if the twin-ratio is close to 0.5.
Q77:
Does SHELXL calculate estimated observed F-values for a single twin component ?
George:
For the purpose of calculating a difference electron density map and the
final Fo/Fc tables (but not of course for refinement) SHELXL partitions
the observed intensity in the ratio of the calculated contributions, i.e.
Fo^2 (for component 1) = Fo^2 (total) * Fc^2 (component 1) / Fc^2 (sum of
all components)
where these Fc values include the twin scale factors k.
This is the weakest link in the treatment of twins and if you have a
better idea please let me know!
Q78:
Where can I read about twinning ?