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--- crystallography:collection:frx:usage [2016/05/23 12:27] – bonnefon
+++ crystallography:collection:frx:usage [2023/11/01 20:19] (current) – external edit 127.0.0.1
@@ Line 1: / Line 1: @@
-==== Rigaku FR-X Usage ====
+====== Rigaku FR-X Usage ======
 Training on 2015.03.30
-FR-X generator: 45 kV, 66 mA, 70 μ cross-section, 180 μ beam, 1.7 miliradians divergence
+**FR-X generator**:
+  * 45 kV,
+  * 66 mA,
+  * 70 μ cross-section,
+  * 180 μ beam,
+  * 1.7 miliradians divergence
-Pilatus 300K detector: 3 chips, shorter exposure time, no readout (no shutter closing), almost never overloaded (>1.5 millions), water circulation at room temperature, dry air
+**XG Control** tells you the settings values (45 kV and 66 mA vs 20 kV and 10 mA when not used) .
+You can screen as soon as the parameters have reached the target values, but wait one more hour to collect, so that the beam is stable.
+**Pilatus 300K detector**:
+  * 3 chips,
+  * shorter exposure time,
+  * no readout (no shutter closing),
+  * almost never overloaded (>1.5 millions),
+  * water circulation at room temperature,
+  * dry air
 <code>>thread</code> command on camserver gives the temperature and humidity level
@@ Line 12: / Line 26: @@
 Cryostream should be at 100 K
-Close 3 glass doors, push the green light on the left
+Close 3 glass doors, push the green light on the left ((HKL does get info on the shutter status))
-Run HKL_3000_P300KChi (the other HKL_3000 version is for data integration only)
+Run HKL_3000_P300KChi ((the other HKL_3000 version is for data integration only))
 <q>Collect/Connect to initialize</q> (also open an instrument server window in the background)
@@ Line 27: / Line 41: @@
 Set the directory /data/user/project1_crystal1
-**dtdisplay**
+==== dtdisplay ====
   * middle click > zoom area
   * middle click > reset zoom
@@ Line 37: / Line 52: @@
 ----
-**Index**
+==== Index ====
   * Refine in triclinic (P1)
   * Fit Basic
   * Fit All
   * Mosaicity (by default it cannot estimate well the mosaicity((default value 0.3)), so you need to select)
+  * With a 3D Window >1, the mosaicity option with already be checked
   * To process up to the corner, you need to index up to the corner!
 ----
-**Strategy**
+==== Strategy ====
   * HKL_3000 strategy only works for single axis
   * Rigaku_Strategy is specially made for FR-X on the 4 axis
@@ Line 61: / Line 79: @@
 For some pins, Chi 60° may be too high and should be restricted to 50°
-Close the window and choose <q>Setup DC</q> to transfer the informations to HKL data collection window
+Close the window and choose <q>Setup DC</q> to transfer the information to HKL data collection window
 <wrap em>Exposure time is not imported from the strategy and should adjusted manually</wrap>
+multi mode is useful to find the best detector distance and exposure time
+Check diffraction images
+go to the data directory
+>dtdisplayhkl *.img &
+to 50 strong reflections are needed for indexing.
+If it is not the case, increase the exposure time.
+It's possible to index non consecutive images if they have the same scan id
+At 2θ ω goes from -90° to +90°, so don't put 90° step with 0° as ω start
+Open display, do the peak search there, frame ↑ (middle click), the peak search has automatically changed, OK
+Chi geometry goniometer, omega angle 180° follows the 2θ angle
+If you press ABORT, the connexion will be lost and you will need to re-initialize
+Check the log on the MSCservdect window.
+To resume, check the parameters in the display.
+The scan number will be implemented.
+With a 3D window >1, the mosaicity box will automatically be checked
+----
+==== Zoom window ====
+**Box size**: (16 by default) usually not necessary to change, even if there are neighbor spots in the box
+Int_box: red ellipsoid to integrate spot. The reject area is between the red and the white. Is it necessary to increase the size for the red ellipsoid (0.35 by default)?
+Changing box size also changes the spot size. Use the box size that gives the highest resolution for the red area (it depends if the number is odd or even).
+**Profile fitting radius** is the yellow circle. When ON, it display only the strongest reflexions. Increase (from 8 to 10) when there are less spots to have enough strong reflexions within the circle, otherwise HKL might reject too many weak reflexions. Don't use a too big radius because spot profile is different at the center and in the edges.
+**3D window**: number of consecutive images put together to estimate the mosaicity. Usually it covers the spread of the mosaicity ONCE, but set it to cover it TWICE.
+**Set shadow region**: the Mask for the beamstop is not critical.
+----
+==== Integrate ====
+All scans are processed
+χ² should be close to 1
+----
+==== Scale ====
+**Check the boxes**:
+  * Scale restrains
+  * Absorption correction
+  * Anomalous
+  * B restrains (HKL recommended)
+Have low reflexions marked for rejection.
+Scale several times until the number don't change anymore.
+If χ² is too low, decrease the error scale factor.
+Exclude frames if necessary.
+**For SAD phasing**:
+if χ² is high: increase the scale factor (up to 1.6) and the error model (up to 0.6-0.7), but it might kill the anomalous signal.
+Check the orange(merged)/blue(unmerged) plot on χ² : higher signal means higher gap
+**Use auto correction**: will try to reach a χ² of 1, but we don't know how, so be careful.
+----
+==== Report ====
+The log file contains merged I+/I- an the end of the file and tables with unmerged I+/I- in the middle of the file.
+----
+==== SAD-Phasing ====
+The sequence is necessary. Give the source or the signal or define if you have a heavy atom.
+Software used: Arp/Warp, CCP4-6.3.0, CCP4-6.4.0(Coot), ShelX
+**Data analysis**:
+Grey area 1.3 for anomalous signal (can be used up to 1.0)
+**Find sites**:
+Choose the resolution limit (>1.8 Å to distinguish disulfide bonds, test 0.1 Å increments)
+Check the absolute value and the spread of solutions (a few in the up-right corner)
+**Site view**:
+Sphere size correlates with the occupancy
+**Phase**:
+High resolution limit; try different resolution cutoffs
+**Check hand** (//ShelXE//):
+You should see the difference, dark red/dark blue, difference of one graduation in scale.
+If necessary, increase the cycle number so that a plateau is reached.
+**Phase**:
+//Mlphase//, //DM-phase extension//
+**NCS**:
+//DM// or //Parrot// (a bit better)
+At least try it if you have more than 1 molecule in the Asymmetric Unit
+**Build**:
+//ARP/WARP// for high resolution
+//Buccaner// for lower resolution (∼ 3 Å)
+Let ARP/WARP build/refine several times if has difficulties building. Start several times witha few cycles rather that once with many cycles.
+----
+==== Molecular Replacement ====
+**Analyze data**
+**Prepare Model**: Edit the PDB file (delete ligands, waters, side-chains if you want)
+**Run MR** (//MolRep//): 3 Å by default; rigid body at 3.0 Å, then refine at max resolution