Results tagged “PX” from The SIBYLS Beamline
A frustrating challenge faced by crystallographers is the often futile and time consuming effort spent improving the resolution of poorly diffracting crystals for complexes. Most efforts to improve diffraction are carried out in the wet lab and entail modifications to sample preparation, crystal growth, and crystal harvesting. Currently, there are few technologies available to improve crystal diffraction quality at the synchrotron. However, the solvent content in protein crystals varies from 30% to 70%, and experiments have shown that humidity changes are correlated with defined (and reversible) unit cell adjustments. Robert Huber’s group developed a revolutionary technology for MX: the Free Mounting Device (FMD), which can manipulate the humidity environment around protein crystals in a controlled and reproducible manner and then allow measurement of diffraction quality as a function of humidity 1. The SIBYLS beamline has the first publicly available FMD system in the world. An example of a result made possible by the FMD was the determination of the crystal structure of the catalytic α subunit of E. Coli DNA polymerase III to 2.3Å resolution2. The previous best resolution was ~3Å and contained disordered domains which became ordered upon dehydration.
FMD - Main Humidty Control Unit
FMD - “Iron Maiden”
Here is the FMD Iron Maiden on the microscope stand, ready for crystal harvesting.
… and on the goniometer.
FMD - Control Software
This is a screenshot of the software for programming in precise humidty ramps.
footnotes
- Kiefersauer R, Than ME, Dobbek H, Gremer L, Melero M, Strobl S, Dias JM, Soulimane T, and Huber R “A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes” J. Appl. Cryst. (2000). 33, 1223-1230
- Lamers MH, Georgescu RE, Lee SG, O’Donnell M, Kuriyan J “Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III” Cell. 2006 Sep 8;126(5):881-92.
A paper has been published in The October 2 issue of Cell by Scott Williams et al. that sheds light on a previously missing piece of the double-strand break repair complex MRN (aka Mre11-Rad50-Nbs1). The paper entitled “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” presents a compelling model of the role of Nbs1 (i.e “N” of the MRN) in coordinating double-strand break processing and repair. The paper was made possible in part by crystal structures and SAXS data of Nbs1 that were collected at the SIBYLS beamline.
Abstract:
The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.
Abstract:Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.
Yeah!
Come collect data on our newly upgraded ADSC Q315r area detector.
Here is the "proof"
DOMO (Dynamic Offsite MX(Macromolecular Crystallography) Operator) mounted crystals remotely for the first time today. The user accessed the beamline via port 22 (i.e. ssh) from San Diego using NX Client and was able to successfully screen through 50 or so crystals. Although I could have monitored the shift from my office, from home, or from Tahiti, I thought it would be more prudent to remain close to the beamline in the unlikely event that intervention was needed…. Well I’m glad that I stuck around. Although DOMO performed well, there were a couple of times that I needed to step in a make things right. Overall, however, it was a very successful first run.
If you are interested in collecting crystallographic data from the comfort of your own lab (or Tahiti) I would encourage you to apply for time on the SIBYLS beamline. We have a small supply of sample cassettes and the necessary tools for loading the cassettes. A cassette kit can be sent to you, whereupon you load the cassette with your valuable samples, and thusly send it back to the beamline in time for your shift. It’s loads of fun!
K-level and L-level emission lines in KeV
No. Element Kα1 Kα2 Kβ1 Lα1 Lα2 Lβ1 Lβ2 Lγ1 3 Li 0.0543 4 Be 0.1085 5 B 0.1833 6 C 0.277 7 N 0.3924 8 O 0.5249 9 F 0.6768 10 Ne 0.8486 0.8486 11 Na 1.04098 1.04098 1.0711 12 Mg 1.25360 1.25360 1.3022 13 Al 1.48670 1.48627 1.55745 14 Si 1.73998 1.73938 1.83594 15 P 2.0137 2.0127 2.1391 16 S 2.30784 2.30664 2.46404 17 Cl 2.62239 2.62078 2.8156 18 Ar 2.95770 2.95563 3.1905 19 K 3.3138 3.3111 3.5896 20 Ca 3.69168 3.68809 4.0127 0.3413 0.3413 0.3449 21 Sc 4.0906 4.0861 4.4605 0.3954 0.3954 0.3996 22 Ti 4.51084 4.50486 4.93181 0.4522 0.4522 0.4584 23 V 4.95220 4.94464 5.42729 0.5113 0.5113 0.5192 24 Cr 5.41472 5.405509 5.94671 0.5728 0.5728 0.5828 25 Mn 5.89875 5.88765 6.49045 0.6374 0.6374 0.6488 26 Fe 6.40384 6.39084 7.05798 0.7050 0.7050 0.7185 27 Co 6.93032 6.91530 7.64943 0.7762 0.7762 0.7914 28 Ni 7.47815 7.46089 8.26466 0.8515 0.8515 0.8688 29 Cu 8.04778 8.02783 8.90529 0.9297 0.9297 0.9498 30 Zn 8.63886 8.61578 9.5720 1.0117 1.0117 1.0347 31 Ga 9.25174 9.22482 10.2642 1.09792 1.09792 1.1248 32 Ge 9.88642 9.85532 10.9821 1.18800 1.18800 1.2185 33 As 10.54372 10.50799 11.7262 1.2820 1.2820 1.3170 34 Se 11.2224 11.1814 12.4959 1.37910 1.37910 1.41923 35 Br 11.9242 11.8776 13.2914 1.48043 1.48043 1.52590 36 Kr 12.649 12.598 14.112 1.5860 1.5860 1.6366 37 Rb 13.3953 13.3358 14.9613 1.69413 1.69256 1.75217 38 Sr 14.1650 14.0979 15.8357 1.80656 1.80474 1.87172 39 Y 14.9584 14.8829 16.7378 1.92256 1.92047 1.99584 40 Zr 15.7751 15.6909 17.6678 2.04236 2.0399 2.1244 2.2194 2.3027 41 Nb 16.6151 16.5210 18.6225 2.16589 2.1630 2.2574 2.3670 2.4618 42 Mo 17.47934 17.3743 19.6083 2.29316 2.28985 2.39481 2.5183 2.6235 43 Tc 18.3671 18.2508 20.619 2.4240 - 2.5368 - - 44 Ru 19.2792 19.1504 21.6568 2.55855 2.55431 2.68323 2.8360 2.9645 45 Rh 20.2161 20.0737 22.7236 2.69674 2.69205 2.83441 3.0013 3.1438 46 Pd 21.1771 21.0201 23.8187 2.83861 2.83325 2.99022 3.17179 3.3287 47 Ag 22.16292 21.9903 24.9424 2.98431 2.97821 3.15094 3.34781 3.51959 48 Cd 23.1736 22.9841 26.0955 3.13373 3.12691 3.31657 3.52812 3.71686 49 In 24.2097 24.0020 27.2759 3.28694 3.27929 3.48721 3.71381 3.92081 50 Sn 25.2713 25.0440 28.4860 3.44398 3.43542 3.66280 3.90486 4.13112 51 Sb 26.3591 26.1108 29.7256 3.60472 3.59532 3.84357 4.10078 4.34779 52 Te 27.4723 27.2017 30.9957 3.76933 3.7588 4.02958 4.3017 4.5709 53 I 28.6120 28.3172 32.2947 3.93765 3.92604 4.22072 4.5075 4.8009 54 Xe 29.779 29.458 33.624 4.1099 - - - - 55 Cs 30.9728 30.6251 34.9869 4.2865 4.2722 4.6198 4.9359 5.2804 56 Ba 32.1936 31.8171 36.3782 4.46626 4.45090 4.82753 5.1565 5.5311 57 La 33.4418 33.0341 37.8010 4.65097 4.63423 5.0421 5.3835 5.7885 58 Ce 34.7197 34.2789 39.2573 4.8402 4.8230 5.2622 5.6134 6.052 59 Pr 36.0263 35.5502 40.7482 5.0337 5.0135 5.4889 5.850 6.3221 60 Nd 37.3610 36.8474 42.2713 5.2304 5.2077 5.7216 6.0894 6.6021 61 Pm 38.7247 38.1712 43.826 5.4325 5.4078 5.961 6.339 6.892 62 Sm 40.1181 39.5224 45.413 5.6361 5.6090 6.2051 6.586 7.178 63 Eu 41.5422 40.9019 47.0379 5.8457 5.8166 6.4564 6.8432 7.4803 64 Gd 42.9962 42.3089 48.697 6.0572 6.0250 6.7132 7.1028 7.7858 65 Tb 44.4816 43.7441 50.382 6.2728 6.2380 6.978 7.3667 8.102 66 Dy 45.9984 45.2078 52.119 6.4952 6.4577 7.2477 7.6357 8.4188 67 Ho 47.5467 46.6997 53.877 6.7198 6.6795 7.5253 7.911 8.747 68 Er 49.1277 48.2211 55.681 6.9487 6.9050 7.8109 8.1890 9.089 69 Tm 50.7416 49.7726 57.517 7.1799 7.1331 8.101 8.468 9.426 70 Yb 52.3889 51.3540 59.37 7.4156 7.3673 8.4018 8.7S88 9.7801 71 Lu 54.0698 52.9650 61.283 7.6555 7.6049 8.7090 9.0489 10.1434 72 Hf 55.7902 54.6114 63.234 7.8990 7.8446 9.0227 9.3473 10.5158 73 Ta 57.532 56.277 65.223 8.1461 8.0879 9.3431 9.6518 10.8952 74 W 59.31824 57.9817 67.2443 8.3976 8.3352 9.67235 9.9615 11.2859 75 Re 61.1403 59.7179 69.310 8.6525 8.5862 10.0100 10.2752 11.6854 76 Os 63.0005 61.4867 71.413 8.9117 8.8410 10.3553 10.5985 12.0953 77 Ir 64.8956 63.2867 73.5608 9.1751 9.0995 10.7083 10.9203 12.5126 78 Pt 66.832 65.112 75.748 9.4423 9.3618 11.0707 11.2505 12.9420 79 Au 68.8037 66.9895 77.984 9.7133 9.6280 11.4423 11.5847 13.3817 80 Hg 70.819 68.895 80.253 9.9888 9.8976 11.8226 11.9241 13.8301 81 Tl 72.8715 70.8319 82.576 10.2685 10.1728 12.2133 12.2715 14.2915 82 Pb 74.9694 72.8042 84.936 10.5515 10.4495 12.6137 12.6226 14.7644 83 Bi 77.1079 74.8148 87.343 10.8388 10.73091 13.0235 12.9799 15.2477 84 Po 79.290 76.862 89.80 11.1308 11.0158 13.447 13.3404 15.744 8S At 81.52 78.95 92.30 11.4268 11.3048 13.876 - 16.251 86 Rn 83.78 81.07 94.87 11.7270 11.5979 14.316 - 16.770 87 Fr 86.10 83.23 97.47 12.0313 11.8950 14.770 14.45 17.303 88 Ra 88.47 85.43 100.13 12.3397 12.1962 15.2358 14.8414 17.849 89 Ac 90.884 87.67 102.85 12.6520 12.5008 15.713 - 18.408 90 Th 93.350 89.953 105.609 12.9687 12.8096 16.2022 15.6237 18.9825 91 Pa 95.868 92.287 108.427 13.2907 13.1222 16.702 16.024 19.568 92 U 98.439 94.665 111.300 13.6147 13.4388 17.2200 16.4283 20.1671 93 Np - - - 13.9441 13.7597 17.7502 16.8400 20.7848 94 Pu - - - 14.2786 14.0842 18.2937 17.2553 21.4173 95 Am - - - 14.6172 14.4119 18.8520 17.6765 22.0652
Values are from J. A. Bearden, “X-Ray Wavelengths”, Review of Modern Physics, (January 1967) pp. 86-99, unless otherwise noted.
Eight easy steps to connect to the SIBYLS NX server through the beamline firewall. These instructions will work for OS X and Linux. We've also compiled more detailed instructions when connecting from a Windows PC.
1. Download and Install NoMachine NX Client.
2. Launch the NX Client and create a new connection.
name it whatever you want
Host: kona.als.lbl.gov
Protocol: SSH
Port: 22
3. Under the Advanced Settings check the "Use the NoMachine login" box.
4. Use your SIBYLS computer account username and password. When you connect for the first time I believe you will be prompted to accept the server key. Just click "yes".
5. you will then need to create a new desktop session. Please select Gnome Desktop. We don't have any of the other window managers installed.
6. You are now connected to kona and should have a desktop available to you. You may want to make it fullscreen and set the 1:1 pixel option to make the best use of your monitor.
The detector can be brought up to essentially room temperature (in order to gracefully shut it down or do other work on it) or brought down to -40°C (to minimize detector noise for data collection) using the “Quantum Console” program on the detector computers.
This figure shows a fairly typical screen image on the detector computers.
In the image shown, the “REMOTE Detector OP” program is already running (as a DOS window). If this program is not running, it needs to be run before the Quantum Console can control the detector modules. The shortcut to the program is highlighted by one of the white circles.
Once the “REMOTE Detector OP” program, hereafter the detector program, is running on all nine modules, you can control the detector temperature by running the Quantum Console program on any one of the detector computers. The shortcut for this program is highlighted by the other white circle.
When the Quantum Console starts, it will open a window that looks something like this:
Before you can do anything else, you need to click on “Connect to Detector Processes.” This will create a row of status reports and allow you to click on “ENABLE Temperature Control”, resulting in a window something like this one:
This window actually shows a temperature-change operation that is already under way, but all the essential parts are visible.
If all is as it should be, setting the temperature is fairly simple. To cool the detector down for operation, click “Ramp to Cold Operating Temp”. To warm it up, click on “Warm up Detector to +10°C”. Either one will gradually bring the detector to the desired temperature. Shifting by the whole range from +10°C to -45°C, in either direction, generally takes about an hour.
Unfortunately, it is occasionally necessary to restart the ADSC detector along with the associated software. This will summarize how that is done. We will assume that everything is up and running at the beginning of the process; starting when some component or other is down is essentially the same.
We’ll assume that this is only a short interruption and that we don’t have to worry about the detector vacuum.
Begin by bringing the detector to room temperature (or +10C) as described in “Detector Temperature Settings.”
Once the detector is warmed up, it can be shut down at the power switches —- it is not necessary to shut down the detector processes or other software first.
Turn the detector power switches (the green switches highlighted by the arrow) off from top to bottom. The main power switches should be turned on starting with the bottom power module, then the middle module, then the top module. The order does matter.
If you don’t have the “REMOTE Detector OP” processes running yet, this is the time to start them. Double click on the “REMOTE Detector OP” shortcut, and you should see a DOS window appear:
Even if you already had these processes running when you restarted the detector, they still must be sent a reset signal. We generally do this from a script. The script makes up part of /programs/beamline/nuke, so it can be found there, but for completeness, here is the one we’ve been using:
foreach module ( detector0 detector1 detector2 detector3 detector4 detector5 detector6 detector7 detector8 )
# send the reset signal
echo -n "$module restart "
echo "restart" | sock_exchange.tcl $module 8038 1
echo ""
end
Entering that script at a shell prompt at any of the BL12.3.1 Linux consoles should reset the whole group of detectors. (Since the BLU-ICE/DCSS control programs run on dataserver, that is a good place to run the script.) This reset signal will cause lots of activity on the “REMOTE Detector OP” windows:
Beamline Diagram
Focus Beam at Beamstop with Experimental Table Between PX and SAXS
Check Helium tank outside of hutch to make sure it is full and that it is flowing into the shutter box. It is important to purge all air from the shutterbox as the ion-guage needs to report a consistent value.
Turn Helium on as early as possible to assure shutterbox is purged
In a terminal window turn off the feedback with the following command:
feedback.com off
Switch KVM to the Beamline computer, and launch "Shortcut to BL Control Main".
Click the white arrow in upper left of main window. This will open the "Beamline 12.3.1 Beamline Control System" window.
Open bothe the Motor Debugger and Motor Monitor modules from the "Motors" pulldown menu.
Turn on autoscaling under the "Amplifiers" pulldown main menu. (why?)
Set M2 Bend Up to 244000, and click move button.
Set M2 Bend Down to 267000, and click move button.
Change "Mono eV" to 10000.
Remove the safety pin and use the hand crank to move the table to the mid point. There is a piece of red tape marked in pen "YAG" indicating the proper midpoint position of the table.
Attach the BNC cable to the camera that monitors the YAG prism, and focus the camera (this step will hopefully be unnecessary once a more permanent camera position is established.
Close the hutch.
Open the main shutter. You should see the direct beam hitting the YAG prism at this point.
Select "tune rocking curve" and click either the step ⬆ or the step ⬇ button once to initialize the optimization procedure. (this automagically adjusts Theta2)
Select "M2 Tilt" and click the home button to reset all values to 0.000
Move M2 Tilt to 12000.
Some PX users will have adjusted the Slits1 to make a very tight beam so they should checked and backed off if necessary. Aperture Line 1 and Aperture Line 11 values should be decreased several unit values.
If you lose the beam jog M2 Tilt in 100 unit increments (this should move the beam up and down on the video monitor).
Select "Chi2" (value should be ~0.492) jog Chi2 in 0.01 increments. (this should move the beam left and right on the video monitor). Because Chi2 is adjusting the focus of the beam changes to this value will drastically alter the shape of the beam.
Sometimes the table will need to be moved slightly in order to position the beam in the middle of the YAG. Additionally the shutter control cable may sometimes get in the way so it must be unplugged from the shutterbox. Turn off power on shutter control box and unplug cable.
Close hutch and turn off hutch light.
Open main shutter and make sure video monitor is turned on.
Optimize the shape and size of the beam. You want it as round and small as possible. Make small adjustments to both Chi2 and M2 Tilt.
Move Experimental Table to SAXS Position
Move the table all the way into SAXS mode using the hand crank. There is a digital dial attached to the air table's sub-frame. Crank the table until this value reads zero.
Re-attach the shutter control cable to the shutter box and turn on the power to the shutter control box. Press the small red button on the shutter control box to really turn it back on.
Close hutch and open main shutter.
Optimize Slits 1, Slits 2, and Slits3 - itertatively
Make sure that the video signal going to the beamline computer is displaying the Beam Position Monitor (BPM) video-feed from the camera that points into the shutter box.
Turn on feedback with the following command:
feedback.com on
The point here is to move slits1 (Aperture guys) and slits2 (SAXS aperture guys) and slits3 (Guard Slits guys) back in as far as possible without actually clipping the beam.
The beam on the back of the shutter may be a bit rough on the bottom edge as a result of nicking slits2 and may look like this:
Jog M2 Tilt to move the whole beam slightly up.
Make sure the High Voltage control unit that feeds the ion guage in the shutterbox has not been tripped. If it has been tripped then reset and increase voltage to as close to 300V as possible.
Close all four slits1 blades as far as possible without clipping beam.
Aperture Line 1moves in from the rightAperture Line 11moves in from the leftAperture Uppermoves in from the top... duhAperture Lowermoves in from the bottom... duh
Close all four slits2 blades as far as possible without clipping beam.
SAXS Aperture Line 1moves in from the rightSAXS Aperture Line 11moves in from the leftSAXS Aperture Uppermoves in from the bottom... huh?SAXS Aperture Lowermoves in from the top... huh?
Insert all filters into direct beam and take a 1sec shot on the MAR CCD.
Close all four slits3 blades as far as possible without clipping beam.
Guard Slits 1more + values to close blade down. (Left side)Guard Slits 11more - values to close blade down. (Right side)Guard Slits Uppermore + values to close blade down.Guard Slits Lowermore - values to close blade down.
Reduce Reflections from Slits3 and adjust beamstop
Take a 1 sec shot on MAR CCD. It will look something like this:
Back off the Guard Slits Upper and Guard Slits Lower until the vertical streaking is eliminated. (streaking toward the top of the screen is caused by Guard Slits Lower being too far in, and vice-a versa for streaking downwards)
Back off the Guard Slits 1 and Guard Slits 11 until the horizontal streaking is eliminated. (this is usually less of a problem, but again streaking to the left is caused by x-rays reflecting off of Guard Slits 11, and vice-a-versa for streaking to the right)
Adjust Endstop y to move the beamstop up and down.
Adjust Endstop x to move the beamstop left and right.
Center the beamstop by observing a line integration tool drawn though the beamstop. The idea is to get a symmetrical background scatter aboce and below the beamstop shadow.
Define Beam for Users
Enter hutch. Manually open the experimental shutter. Insert CCD shield. Insert sample cell with fluorescent paper.
Close hutch. Open main shutter and define the bounding box for the beam on the video monitor.
It may be necessary to move the sample up or down (Sample x and Sample y) so that the beam enters the sample cell in the center.
Enter hutch. Close experimental shutter. Remove CCD shield.
You are now ready to CRUSH!!!!!
A new review on macromolecular SAXS has been published in the Quarterly Reviews in Biophysics by Putnam, C.D., Hammel, M., Hura, G.L., and Tainer, J.A.
“This six part review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures.”
The review provides an extensive and up-to-date review on the application of small angle X-ray scattering is available for download
.
Quick Links
