Cryostream alignment

 

  • The distance between the nozzle and the sample should be less than 5 mm.
    • If necessary, adjust with the translation screw on the cryostream holder.
    • A good way to check the distance is to change to zoomlevel 1, and then move the cryostream as close as possible, but such that it does just not show up in the video image.

 

  • Quick visual check whether the cryostream is correctly aligned laterally:
    • Center a sample in mxcube.
    • Go to the hutch and check that the sample is exactly on-axis with the center of the cryo nozzle
      (i.e., within about 1 mm of the nozzle axis).
    • Do so from two viewpoints that are 90° apart, and you are sure that the cryostream is well aligned laterally.

 

 

Note: Please be careful not to touch the beamstop when moving the cryostream!

In particular, please check the translation of the cryostream to make sure it is in the "in" position.
If you align the cryostream while it is in the "out" position, moving it back to the "in" position will slam it into the beamstop etc.

(xxx how to control or move it?? xxx)

 

 

Aligment procedure

 

  • You may want to use the alignment tool: a metal piece about 1 cm in diameter, with a needle in the middle.
  • Find it somewhere on the beamline (e.g., attached to the cryostream support).
  • In mxcube, move the beamstop to the "out" position.
  • Place the alignment tool on the nozzle of the cryostream. (This is OK to do while it is running.)
  • Mount a sample pin and center it in mxcube (autocentring is good enough).
  • Carefully move the cryostream so that the tip of the alignment tool just (not) touches the tip of the sample pin.
  • Remove the alignment tool. 
  • Check again that the cryostream is in the "in" position.
  • Move the beamstop back in (with mxcube) and check that it does not collide with the cryostream.

 

 

Importance of cryostream distance from sample

Test data were collected from a single trypsin crystal supported in a nylon loop with the cryostream aligned on the sample but the cryostream at 3 different distances from the sample. Four 180° (multiplicity ~7) data sets were collected with 1° oscillations, 3 passes, 1 second exposures with cryostream to sample distances of 5 mm, 10 mm, 15 mm and 5 mm again (to account for radiation damage, etc).

We do not observe such marked effects when using litholoops.

Two datasets were also collected at 5 mm and 15 mm cryostream-to-sample distances with 10 s exposures with 10 passes with the beam attenuated to approximately a tenth of the prior data sets.

 

Conclusios: it is advisable to place the cryostream to within 5 mm of the sample while collecting data (and certainly closer than 10 mm).

 


...and here are the numbers:



-----------------------------------
-mattset 1; 1o 1sec 3 passes; 180o-
-----------------------------------

############################
# Cryostream 5mm from xtal #
############################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.027  0.024  0.027  0.013    562    6758.   312.7  21.6   348. 47.3    4974    804   242  -0.028   1140
  2 0.0692  3.80 0.026  0.022  0.026  0.013   1153   13651.   637.3  21.4   692. 50.6    9606   1432   279  -0.032   2326
  3 0.1038  3.10 0.027  0.023  0.027  0.011   1536   11393.   540.8  21.1   573. 51.8   12732   1823   287  -0.030   3058
  4 0.1384  2.69 0.029  0.025  0.027  0.012   1834    5987.   299.5  20.0   304. 50.5   15100   2119   285  -0.030   3585
  5 0.1730  2.40 0.030  0.026  0.028  0.014   2103    4312.   216.2  19.9   221. 49.1   17207   2395   292  -0.027   4157
  6 0.2076  2.19 0.031  0.027  0.028  0.013   2353    3745.   187.4  20.0   196. 47.9   19106   2642   289  -0.028   4484
  7 0.2422  2.03 0.034  0.031  0.029  0.015   2556    3256.   179.3  18.2   183. 44.2   20589   2837   281  -0.031   4837
  8 0.2768  1.90 0.038  0.034  0.030  0.016   2777    2438.   143.0  17.1   145. 41.1   22325   3064   284  -0.029   5313
  9 0.3114  1.79 0.045  0.040  0.030  0.018   2934    1605.   109.1  14.7   103. 36.5   23452   3221   282  -0.027   5589
 10 0.3460  1.70 0.053  0.048  0.031  0.023   3112    1213.    95.6  12.7    83. 33.6   24514   3388   271  -0.028   5748
 Overall:        0.031  0.027  0.031  0.014  20920    4332.   272.2  15.9   232. 43.8  169605  23725  2792  -0.030  40237
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias

#############################
# Cryostream 10mm from xtal #
#############################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.037  0.033  0.037  0.018    563    6509.   456.2  14.3   410. 37.2    5037    807   245  -0.035   1117
  2 0.0692  3.80 0.038  0.034  0.038  0.016   1156   13176.   937.6  14.1   812. 40.2    9769   1438   284  -0.036   2333
  3 0.1038  3.10 0.037  0.034  0.037  0.017   1535   10786.   750.8  14.4   659. 41.3   12814   1822   287  -0.032   3097
  4 0.1384  2.69 0.038  0.035  0.038  0.016   1836    5617.   395.7  14.2   346. 40.3   15182   2123   287  -0.030   3558
  5 0.1730  2.40 0.040  0.036  0.038  0.017   2103    4030.   291.5  13.8   251. 39.3   17250   2394   291  -0.034   4149
  6 0.2076  2.19 0.040  0.036  0.038  0.017   2353    3494.   232.8  15.0   221. 38.4   19165   2643   290  -0.029   4500
  7 0.2422  2.03 0.044  0.040  0.039  0.019   2561    3019.   228.4  13.2   205. 35.7   20655   2842   281  -0.030   4857
  8 0.2768  1.90 0.048  0.044  0.040  0.020   2776    2261.   178.0  12.7   162. 33.3   22315   3063   284  -0.025   5285
  9 0.3114  1.79 0.056  0.050  0.040  0.023   2935    1482.   131.4  11.3   115. 29.5   23456   3221   281  -0.020   5531
 10 0.3460  1.70 0.064  0.058  0.041  0.027   3112    1116.   109.6  10.2    94. 27.1   24521   3389   272  -0.022   5811
 Overall:        0.041  0.037  0.041  0.018  20930    4093.   377.3  10.8   265. 35.1  170164  23742  2802  -0.031  40238
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias

#############################
# Cryostream 15mm from xtal #
#############################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.100  0.097  0.100  0.045    565    7744.  1486.5   5.2   916. 19.6    5057    811   246  -0.038   1228
  2 0.0692  3.80 0.099  0.094  0.099  0.046   1159   15371.  2952.5   5.2  1805. 21.0    9797   1445   286  -0.028   2481
  3 0.1038  3.10 0.099  0.093  0.099  0.043   1538   12552.  2317.6   5.4  1474. 21.3   12825   1825   287  -0.034   3292
  4 0.1384  2.69 0.097  0.093  0.098  0.044   1836    6465.  1166.8   5.5   768. 20.7   15166   2123   287  -0.041   3850
  5 0.1730  2.40 0.097  0.093  0.098  0.042   2111    4604.   824.1   5.6   553. 20.1   17313   2404   293  -0.026   4550
  6 0.2076  2.19 0.098  0.093  0.098  0.042   2353    3956.   713.4   5.5   485. 19.6   19141   2644   291  -0.028   4816
  7 0.2422  2.03 0.100  0.095  0.098  0.043   2562    3441.   629.0   5.5   458. 17.9   20666   2844   282  -0.039   5169
  8 0.2768  1.90 0.101  0.095  0.099  0.043   2783    2560.   469.0   5.5   362. 16.6   22361   3066   280  -0.040   5663
  9 0.3114  1.79 0.107  0.102  0.099  0.046   2941    1654.   322.5   5.1   255. 14.6   23505   3228   282  -0.021   6032
 10 0.3460  1.70 0.111  0.103  0.099  0.050   3117    1244.   243.2   5.1   209. 13.3   24573   3393   270  -0.026   6261
 Overall:        0.099  0.094  0.099  0.044  20965    4706.  1148.1   4.1   589. 17.8  170404  23783  2804  -0.032  43342
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias

##################################
# Cryostream 5mm from xtal again #
##################################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.025  0.022  0.025  0.013    565    6164.   274.4  22.5   307. 47.0    5071    811   246  -0.028   1153
  2 0.0692  3.80 0.025  0.021  0.025  0.011   1158   12361.   565.5  21.9   608. 50.5    9798   1442   287  -0.030   2347
  3 0.1038  3.10 0.025  0.021  0.025  0.010   1536    9951.   431.2  23.1   487. 51.4   12822   1823   287  -0.029   3047
  4 0.1384  2.69 0.027  0.023  0.025  0.011   1838    5094.   241.9  21.1   252. 50.0   15193   2125   287  -0.030   3554
  5 0.1730  2.40 0.029  0.024  0.026  0.012   2105    3635.   175.3  20.7   182. 48.6   17268   2398   293  -0.029   4125
  6 0.2076  2.19 0.030  0.026  0.026  0.013   2356    3119.   149.5  20.9   159. 47.3   19180   2647   291  -0.027   4433
  7 0.2422  2.03 0.034  0.030  0.027  0.015   2563    2698.   144.8  18.6   148. 43.7   20654   2843   280  -0.029   4740
  8 0.2768  1.90 0.040  0.035  0.028  0.016   2776    2000.   122.8  16.3   117. 40.4   22292   3061   282  -0.025   5248
  9 0.3114  1.79 0.049  0.043  0.029  0.020   2939    1304.    94.4  13.8    84. 35.5   23483   3226   282  -0.022   5463
 10 0.3460  1.70 0.059  0.053  0.030  0.025   3114     972.    84.1  11.6    68. 32.2   24504   3392   273  -0.022   5720
 Overall:        0.030  0.026  0.030  0.013  20950    3728.   228.6  16.3   194. 43.1  170265  23768  2808  -0.028  39830
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias

-------------------------------------
-mattset 3; 1o 10sec 10 passes; 180o-
-------------------------------------

############################
# Cryostream 5mm from xtal #
############################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.026  0.024  0.026  0.013    570    3520.   173.1  20.3   182. 45.0    5190    823   253  -0.017   1078
  2 0.0692  3.80 0.025  0.021  0.025  0.010   1162    6832.   325.5  21.0   351. 48.2    9988   1454   292  -0.024   2269
  3 0.1038  3.10 0.024  0.020  0.024  0.010   1534    5344.   223.4  23.9   276. 48.1   12880   1822   288  -0.020   2837
  4 0.1384  2.69 0.026  0.024  0.025  0.011   1838    2687.   118.4  22.7   142. 45.9   15215   2125   287  -0.023   3277
  5 0.1730  2.40 0.030  0.026  0.025  0.012   2106    1902.    88.2  21.6   103. 43.9   17310   2399   293  -0.020   3807
  6 0.2076  2.19 0.032  0.029  0.026  0.013   2358    1623.    79.1  20.5    90. 42.2   19214   2649   291  -0.017   4082
  7 0.2422  2.03 0.038  0.035  0.027  0.016   2559    1397.    80.4  17.4    85. 38.5   20660   2841   282  -0.018   4406
  8 0.2768  1.90 0.048  0.043  0.029  0.019   2783    1030.    71.6  14.4    69. 34.7   22369   3066   280  -0.018   4829
  9 0.3114  1.79 0.064  0.058  0.031  0.026   2937     663.    61.2  10.8    52. 28.9   23490   3225   283  -0.011   5031
 10 0.3460  1.70 0.082  0.074  0.032  0.033   3116     492.    56.3   8.7    44. 25.2   24583   3396   272  -0.012   5310
 Overall:        0.032  0.029  0.032  0.014  20963    1989.   126.6  15.7   113. 38.0  170899  23800  2821  -0.020  36926
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias

#############################
# Cryostream 15mm from xtal #
#############################
   N 1/d^2 Dmin(A) Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias
  1 0.0346  5.38 0.056  0.052  0.056  0.024    571    3762.   392.5   9.6   288. 30.2    5198    825   254  -0.020   1306
  2 0.0692  3.80 0.053  0.050  0.054  0.022   1162    7280.   740.7   9.8   556. 32.4    9982   1453   291  -0.018   2660
  3 0.1038  3.10 0.052  0.048  0.053  0.023   1541    5660.   542.9  10.4   434. 32.4   12924   1830   289  -0.016   3378
  4 0.1384  2.69 0.055  0.051  0.054  0.024   1838    2834.   284.1  10.0   222. 30.8   15210   2125   287  -0.016   3917
  5 0.1730  2.40 0.054  0.051  0.054  0.023   2114    2009.   188.9  10.6   161. 29.4   17352   2406   292  -0.015   4649
  6 0.2076  2.19 0.058  0.054  0.054  0.025   2356    1715.   172.6   9.9   142. 28.2   19191   2648   292  -0.016   4903
  7 0.2422  2.03 0.062  0.058  0.055  0.026   2566    1483.   154.3   9.6   135. 25.6   20721   2848   282  -0.012   5290
  8 0.2768  1.90 0.070  0.064  0.056  0.029   2783    1091.   125.9   8.7   109. 23.0   22381   3068   282  -0.014   5735
  9 0.3114  1.79 0.082  0.074  0.057  0.034   2946     701.    87.4   8.0    82. 19.1   23545   3233   282  -0.009   6232
 10 0.3460  1.70 0.099  0.088  0.059  0.041   3113     521.    76.1   6.8    72. 16.5   24571   3391   271  -0.012   6362
 Overall:        0.059  0.054  0.059  0.025  20990    2109.   282.7   7.5   178. 25.3  171075  23827  2822  -0.016  44432
                  Rmrg  Rfull   Rcum  Ranom  Nanom    Av_I   SIGMA I/sigma  sd Mn(I)/sd Nmeas  Nref  Ncent FRCBIAS  Nbias