A Day in The Life of a User: FED Ids, Pucks and High Hopes

So far most of my posts have focused on the science theory behind X-ray crystallography. But what of the people, both user and staff, who use the beamlines to produce such fanciful structures as this yeast betaprime COP 1-304H6 beta propeller pictured below.

Search 2YNO in the PDB for more details

Upon arriving at diamond its straight to reception to get a site pass and then on to the user office to get your access card for the synchrotron and beamlines, providing you’re not arriving the night before for a shift in the morning; then it’s off to Ridgeway House or another one of the hotels you may be sent to.
Lets assume you all know what staying in a hotel is like and move along to what actually happens at a beamline. First you will meet your friendly neighbourhood beamline scientist who will go through what is new on the beamline, how well the beamline has been operating and then give you a safety induction and some forms to sign. The main component of this is being showed how to search the hutch. For more details on the experimental hutch please see the previous post. It’s a pretty simple search, first swipe your access card, then walk to the back of the hutch, check for any people that may be hiding in the hutch and press a few little white buttons as you go. Make sure not to press the big yellow button pictured below. 

Emergency beam off button: Not to be mistaken for the search buttons. 

Once all of that is out of the way and papers have been signed it’s time to start loading samples. Some users will have samples preloaded in pucks, others will bring them in canes and load them into pucks at the beamline. Either way you will be greeted with a plethora of tools for all your sample loading needs. 

From the bottom going clockwise we have protective gloves to prevent liquid nitrogen burns (not entirely effective), more gloves in latex/ nitrile form, safety glasses, a hair dryer, ethanol, water and compressed air, various tongs and such, the all important blue roll, pipettes and tips, puck tools and finally pucks. 

Start off by filling up some foam dewars (blue and purple things at the bottom of the picture above) from a tipping dewar such as this: 

Tipping dewar

Next cool down your pucks in the liquid nitrogen filled dewar: 

Foam dewar and cooled uni pucks

Transfer your samples on canes from your travel dewar to a tall foam dewar:  

Protein samples on pins in liquid nitrogen filled vials on a cane.

These samples are then placed into pucks and the pucks are then transferred to the large liquid nitrogen dewars in the experimental hutch ready for the robot to load the samples. In the last post I meant to get a robot picture but forgot so here you go. 

Left: beamline set up with goniometer, liquid nitrogen stream etc. Right: Sample handling robot and 2 Liquid nitrogen dewars for sample storage.

Once you’ve got everything loaded into the dewars you do your hutch search just like your friendly neighbourhood beamline scientist taught you and you’re ready to fry some crystals with X-rays. Now all you’ve got to do is remember your FED ID and password which consist of many random letters, numbers and symbols in no particular order and your good to go. Once you’ve inevitably had to scroll through many e-mails to find IDs and passwords it’s time to get comfy because it’s probably going to be a long night. For how every many hours (8, 16 or 24 usually) you’ll be looking at this (see below). Top left screen is the beam status and the all important canteen is open/ closed window. Top right is your last diffraction image and videos of the robot and goniometer to make sure they’re doing what they should be doing. Bottom left is the GDA client where you tell the beamline how many images to take, what sort of rotation, resolution, beam intensity etc. Bottom right, more of a wild card, put what ever you like there but generally its ADXV or ALBULA, 2 programs used to looks at your diffraction patterns up close. 

More screens than you can shake a stick at.

So from here its essentially the same thing all night, you tell the robot to load your sample and then head over to this screen (below) in the GDA where you centre your sample by clicking on the crystal and then rotating 90 degrees and repeating until happy. Then its over to the experimental control where you take 2-3 test shots and a data set if all goes well. 

Sample centring screen: Crystal on a micromount, red circle is where your beam is hitting it, stuff to the left is used for centring. Click away until you’re happy.

Now its just a matter of  refreshing this web page (below) to watch the graphs go up and down until it’s time to load another sample. This can happen anywhere between 8-45 minutes, or more, depending on the beamline you’re on and the number of images you want to collect. 
Now you know what it’s like to spend a day as a crystallographer, but don’t let that deter you. It’s all worth it when you get some good results and a pretty structure to call your own. Or that’s what I’m telling myself until it happens. 
Until next time thanks for reading. 
Sam  http://dlvr.it/31KsT1

Beamlines: Goniometers, Detectors and Comfy Chairs

Hello! Sorry for the massive absence in posting. I’ve been busy/ lacked the motivation to write. From now on I will try my best to post twice a month. Feel free to hurl abuse if I go awol again.

In the last post I gave a brief description of what exactly a synchrotron is and how it works. To the best of my abilities. In this post I’ll be going through where the magic happens, the beamlines. This is where the users, and Diamond staff, work until the early hours of the morning collecting diffraction data from crystals. Unless you’re lucky enough to get a day shift, which I have not this Friday.  
Most beamlines at Diamond are optimised for specific purposes such as micro-focus or tuneable beams, in-situ screening etc, but all have certain things in common. I found this lovely little image on the diamond website to help me guide you through the general world of beamlines. From left to right we have the control cabin where you can sit on your comfy seats, drink coffee, control your data collection and also prepare your samples to be loaded. The next is the experimental hutch where all the cool looking stuff like detectors, goniometers, sample handling robots and the samples you’re testing can be found. This is all encased in a lead shielded box to stop the X-rays getting out. Finally there is the optics hutch. This is where the synchrotron light is filtered and focused, fresh off the storage ring and insertion devices. Again surrounded by lead. Not sure what a storage ring or insertion device is? Please see the previous entry.       
General Layout of a Beamline: Taken From the Diamond Website
Across the whole of the Diamond light Source there are 31 beamlines including macromolecular crystallography, circular dichroism, small molecule crystallography, tomography and many others. Most of these I have never seen, used or have much clue exactly what they are for. So I’ll stick to what I know, that being the 6 macromolecular crystallography (MX) beamlines. As I said before, all MX beamlines have certain common aspects; parts essential to the general function of the beam line such as detectors, goniometers, on axis viewing systems (to see your crystal), liquid nitrogen streams, sample changing robots, sample dewars and lots of wires, panic buttons, safety features and such.
Beamline set up from bottom clockwise: Goniometer, on axis viewing system, cryo stream and sample handling robot.  
IO2 setup from left clockwise: Goniometer, on axis viewing system, de-icing gadget and cryo stream.  
 Safety feature: Only to be pressed if in dire need. The pressing of this will make many people annoyed at you. 
Sample handling robot (left) and sample storage dewars (back and right)
Pilatus detector: Stupid amounts of pixels and money. (not a picture from Dimond)
Having all the beamlines exactly the same would be pretty booring and a little redundant considering the variability of protein crystals. A good example of specialisation to account for crystal type is the I24, or micro focus, beamline. This beamline is specially designed for use with the smallest of protein crystals (10s of microns) as well as long thing rods, utilising its small beam of between 5-30 microns. Many membrane proteins (evil, difficult, horrible proteins to work with) produce these tiny crystals and I24 vastly improves the  chance of determining a structure, generally using multiple crystals. 
If we’re talking about specialised beamlines I should probably mention the one I’m involved with, I23. This is one of the Phase 3 beamlines currently being built at Diamond and is a dedicated phasing beamline utilising long wavelength X-rays to increase the anomalous signal of naturally occuring sulphur atoms in a protein. If you can’t remember what I mean by phasing, please see my previous entry. The use of these longer wavelengths, up to around 4A, present a plethora of challenges and I’m very thankful I’m not an engineer working on them. I may have mentioned before that longer wavelengths means greater absorption. This leads to the whole experiemntal setup being put into a vaccum chamber to reduce absorption in air. This means everything has to be vaccume compatible and reliable enough that you won’t have to break the vaccuum to fix. A tomography system is also in developement to aid in absorption correction. More on that another time. The other major challenge, appart from fitting everything into a smallish space, is how to both mount and cool the samples. As the system is completely in vaccume, standard cooling using a stream of liquid nitrogen is not possible. The Solution to this is cooling by conductance along the pin to the sample, meaning the sample mounts must be both super conducting as well as non-absorbing (pesky long wavelength X-rays). This brings a whole new load of problems as these sample mounts will be very brittle and won’t stand up to the stresses of fishing crystals like other mounts do. As a result we are currently developing a method to transfer samples from nylon loops to these spcial mounts. Amgain, ore on that as the story develops. 
I think I’ve rambled on for long enough now so that is it for this post. I’ll finish with a conceptual picture of the detector specially designed for I23 by Dectris. Look at the size of that beast!

And on that note, see you next time. 
Sam  http://dlvr.it/2wNNwc

Inside the Synchrotron: Magnets, Electrons and Those All Important X-rays

So obviously I’ve mentioned that I work at The Diamond Light Source, the only synchrotron in the UK. From my experience, most people I talk to have no idea Diamond exists let alone what this sci-fi sounding place is and how it works. Building on my experience of telling people what Diamond is and how it works I feel I really need to stress this point. Diamond is not like CERN. I don’t know how CERN works but I do know it and Diamond are worlds apart. They are both circular (technically) and use very powerful bending magnets to steer and focus a beam. But this is the crucial difference, the beams in question are very different and are what makes the experiments performed so profoundly different. 
Anyway, I’m not going to talk about CERN because explaining how that works seems terrifying and I’ve got no clue. Synchrotrons I’m slightly more confident about but I’m still a biologist no a physicist or engineer remember. Lets start with the general structure of a synchrotron, as pictured below. 

Cartoon of the Diamond Light Source Synchrotron. Taken from diamond.ac.uk
Electrons are generated in the electron gun, this is not unlike a giant cathode ray tube from a now obsolete television. Ask your parents, or more likely google, if that’s lost on you. The electrons then pass through 3 particle accelerators in the form of the Linear accelerator (Linac), which accelerates the particles to an energy around 100 MeV using a tuned electric field,  followed by the booster synchrotron which further accelerates the electrons energy to 3 GeV using six dipole bending magnets and a radio frequency voltage source. Finally the electrons pass into the storage ring where, in diamond at least, 48 large electromagnets curve the electrons around the 24 straight sections that make up the 562 m storage ring. Pictured below is the point where the booster synchrotron feeds into the storage ring.  
Inside the storage ring where the booster synchrotron meets the storage ring 
Looking from the outside you could be forgiven for assuming that synchrotrons are circular, but they are in fact polygons made up of multiple straight sections with bending magnets that direct the electrons down these paths. This change in direction causes the electrons to lose energy in the form of light which is then used on the beamlines for experiments. 
 Straight section within the storage ring
A bending magnet (green) and quadrupole magnet (red) 
Quadrupole magnets in red and sextupole magnet in yellow 

Diamond is a third generation synchrotron. This means that in addition to the quadrupole and sextupole magnets pictured above, that vertically and horizontally focus the beam, the storage ring also uses insertion devices. Insertion devices are made up of arrays of magnets that allow the generation of more intense/ bright light (more photons per second) as well as increasing the spectral range and making the beam more tuneable without deflecting or displacing it (sending it flying into a wall or something). These insertion devices come in 2 forms, undulators and wigglers.  

An undulator provides increased brightness within the plan of deflection of the beam to give peaks at 1 or a few wavelengths. This means the beam is bent slightly to produce an electromagnetic wave (yellow wave below) so extra light is given off by the electrons, but not to the point where it deflects like in the bending magnets. This leads to an increase in beam intensity while maintaining a continuous frequency range (green beam below). This continuous frequency range produces a monochromatic beam, or single wavelength/ frequency.  

Radiation produced at an undulator. Taken from the Spring 8 website. 
An undulator (purple) in the Diamond storage ring
A wiggler, much like the undulator, causes the electrons to take a wiggling path instead of their natural straight path. The main difference being that a wiggler produces a beam with a much larger deviation angle to the undulator to produce a much more intense beam over a broader spectrum of X-rays. This gives a higher energy beam with greater penetrating power.

 A wiggler (also purple) in the storage ring: not for the faint hearted, roll snare drum and end of post. 
I hope this has been informative and has made it a little clearer how a synchrotron works. A lot more detail on all of this can be found online, most synchrotron websites have descriptions, pictures and animations usually aimed at a non science reader. Obviously this was just scratching the surface of the physics behind all of this but you’ll have to go elsewhere if you want to learn more. 
If you have questions I will try my very best to answer in the comments. 
Until next time, where I”ll be talking about beamlines, thank you for reading. 
Sam 

   
   http://dlvr.it/2YpqsN

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Blog post number 6: what I did and saw during my trip to The Royal Society for Diamond’s 10th anniversary symposium. The Royal Society MottoThe Royal Society mace

Thermolysin protein crystals. 

For more on X-ray Crystallography and protein crystal please visit http://justanotherphdstudent.blogspot.co.uk/

(Source: justanotherphdstudent.blogspot.co.uk)

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Optimisation of Insulin crystal growth by alteration of the ratios of protein to well solution in the drops. Also why having a beard can cause problems for crystal growth. 

More details at http://justanotherphdstudent.blogspot.co.uk/

(Source: justanotherphdstudent.blogspot.co.uk)

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Glucose Isomerase Crystals before and after Optimization. All that changed was the incubation temperature. More details at http://justanotherphdstudent.blogspot.co.uk/2012/11/crystal-optimization-patience-planning.html

(Source: justanotherphdstudent.blogspot.co.uk)

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Blog post 5 of my times at the Diamond light Source. This week is optimization of crystal growth with more pretty pictures of crystal before and after being optimized. 

Glocose isomerase from 20 degree incubationGlucose Isomerase crystals from 4 degree incubation

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clearscience:

A synchrotron light source is a giant scientific facility made to generate X-rays. We wondered what X-rays could be useful for. It turns out they have just the right wavelength to figure out the location of atoms in a solid material.
X-rays with their wavelengths lined up strike the sample material at some angle, and they get bounced off elastically by the electrons in the atoms. Then you detect the bounced off X-rays at the same angle.
At some special angles, X-rays bouncing off different atoms will overlap, but their wavelengths might not line up anymore. You Clear Scientists know that overlapping light waves interfere with each other. And from this interference, you can use geometry to figure out the atomic spacing (5 nm in this example).

clearscience:

A synchrotron light source is a giant scientific facility made to generate X-rays. We wondered what X-rays could be useful for. It turns out they have just the right wavelength to figure out the location of atoms in a solid material.

X-rays with their wavelengths lined up strike the sample material at some angle, and they get bounced off elastically by the electrons in the atoms. Then you detect the bounced off X-rays at the same angle.

At some special angles, X-rays bouncing off different atoms will overlap, but their wavelengths might not line up anymore. You Clear Scientists know that overlapping light waves interfere with each other. And from this interference, you can use geometry to figure out the atomic spacing (5 nm in this example).

39 notes

cat-powuh:

A crystallography image from a protein.
Crystallography is a lab technique applied to study the structure and composition of biomolecules, it unveiled the structure of hundreds of proteins, and is used until today due to its precision.
Science can be really beautiful sometimes

cat-powuh:

A crystallography image from a protein.

Crystallography is a lab technique applied to study the structure and composition of biomolecules, it unveiled the structure of hundreds of proteins, and is used until today due to its precision.

Science can be really beautiful sometimes

35 notes