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Re: [ccp4bb]: non-specifically bound heavy atom?
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Hi Rui
You didn't say how non-isomorphous the datasets are - that would produce
noisy Pattersons but push up the Riso. Non-specific binding on other hand
wouldn't, because it wouldn't change diffraction amplitudes. I assume
you've tried lots of low and high-resolution cutoffs. Your low resolution
reflexions are probably dump if you collected in-house, while
(incorrectly) strong high-resolution differences would mess it all up too.
If anything, you are MORE likely to see the peak from an anomalous signal
than from isomorphous differences, because isomorphism is perfect. In
fact, you may even be able to tell from anomalous data on your home
source, at CuK wavelength. But then, to be sure, you should record a
touch more than 2 observations per reflexion: 4 per Friedel mate (hkl or
-h-k-l) would be a much better idea. Easy to do, just collect the
"minimum" phi-oscillation twice, once at phi and once at phi+180. Then
you *know* you've recorded both hkl and -h-k-l the same number of times.
Incidentally, that goes for your MAD experiment too - make a point of
collecting every wavelength like that. Better two complete wavelengths
than three incomplete or poorly-redundant ones. And while you're at it,
try to get as much low resolution as you can... it makes a huge
difference for solvent flattening.
Process the data as you go, and calculate Pattersons all the time - if
you're lucky you could see a peak when you're only half-way through the
first wavelength.
Good luck!
Phraenquex
> (There is 1 cys in the 38kD protein.) The Hg data
> are reasonably good to 4A (data statistics see below, 92% complete,
> >60% of the data have more than 2 observations). After
> I scaled the Hg data with the native data set, the overall Riso
> is 18.7% (see below for statistics from scaleit). But the difference
> patterson map does not have any peaks standing out. The highest
> peaks on harker sections are only about 3sigma. I am a little
> puzzled. Somebody has suggested to me that the Hg compound could
> be binding to the protein surface non-specifically, that is why
> I couldn't see any peak in the difference patterson. I am wondering
> if anybody else had similar experience before.
> I greatly appreciate your comments/suggestions on the situation.
> The question following this is: we are going to a synchrotron in a few
> days. I imagine I still will see a Hg peak in the absorption spectrum
> even if it is non-specifically bound. Am I wasting time in trying to
> collect a Hg MAD data set?
>
> Thank you very much for your help.
>
> Sincerely, Rui.
>
> scalepack output:
> Shell Lower Upper Average Average Norm. Linear Square
> limit Angstrom I error stat. Chi**2 R-fac R-fac
> 30.00 8.56 18716.3 586.0 166.3 9.923 0.054 0.070
> 8.56 6.82 5377.2 282.6 126.8 1.600 0.063 0.069
> 6.82 5.96 2321.7 189.7 127.0 1.145 0.084 0.085
> 5.96 5.42 2097.3 205.4 136.1 0.977 0.086 0.078
> 5.42 5.04 2087.1 234.5 145.7 0.821 0.088 0.076
> 5.04 4.74 2233.7 311.6 159.9 0.643 0.096 0.088
> 4.74 4.50 2502.8 366.1 174.0 0.599 0.091 0.086
> 4.50 4.31 2010.1 532.4 186.0 0.400 0.110 0.096
> 4.31 4.14 1407.5 480.6 201.2 0.397 0.148 0.132
> 4.14 4.00 1230.8 386.5 215.3 0.612 0.174 0.152
> All reflections 4013.7 357.4 163.7 1.681 0.077 0.073
>
> Scaleit output:
> <4SSQ/LL> Res NRef <FP**2> Sc_kraut SCALE RFAC RF_I Wted_R <diso> max(diso)
> 0.008 11.3 39 469798. 1.154 1.107 0.204 0.308 0.239 124.1 473
> 0.011 9.6 165 334552. 1.049 1.009 0.191 0.291 0.254 96.9 449
> 0.014 8.4 203 236577. 0.993 0.963 0.183 0.289 0.244 76.7 283
> 0.017 7.6 215 118957. 1.048 1.005 0.205 0.294 0.231 59.8 317
> 0.020 7.0 242 60026. 1.049 1.007 0.202 0.321 0.171 43.1 185
> 0.023 6.5 260 44330. 1.046 0.996 0.209 0.345 0.168 39.0 192
> 0.027 6.1 271 42172. 1.050 1.016 0.189 0.303 0.153 33.8 162
> 0.030 5.8 304 36710. 1.063 1.027 0.189 0.290 0.164 31.3 125
> 0.033 5.5 317 47045. 1.064 1.039 0.165 0.233 0.145 29.7 204
> 0.036 5.3 313 47731. 1.052 1.024 0.181 0.261 0.159 32.3 132
> 0.039 5.1 315 44372. 1.048 1.020 0.175 0.264 0.137 31.4 141
> 0.042 4.9 319 54105. 1.058 1.022 0.189 0.299 0.148 37.0 165
> 0.045 4.7 311 67814. 1.041 1.012 0.173 0.283 0.121 38.2 185
> 0.048 4.5 306 69882. 1.048 1.023 0.167 0.273 0.113 37.2 191
> 0.052 4.4 304 65554. 1.046 1.019 0.175 0.280 0.120 37.7 147
> 0.055 4.3 311 49965. 1.008 0.985 0.176 0.279 0.098 33.9 143
> 0.058 4.2 290 46024. 1.072 1.041 0.202 0.329 0.121 37.4 176
> 0.061 4.1 435 45158. 1.053 1.024 0.203 0.314 0.120 36.9 263
> THE TOTALS 4920. 74753. 1.046 1.012 0.187 0.291 0.169 41.0 473.
>