Here some useful tips from how to get access to the MSKC facility to how to get your crystals to SLAC.
Question: How much protein do I need to set up a crystallization experiment?
Answer: Our Douglas Oryx 8 is capable of nano-liter dispensing, so one typical 96-well screening plate will require ~150 µg of protein (96 x 150 nl @ 10 mg/ml). Therefore, ~1 mg protein will allow the researcher to screen six 96-well crystallizations screens. Each crystallization screen is composed by a mixture of three main components: a precipitant, a buffer, and something else (usually, ions). Along with crystallization cocktails, you might want to test protein concentration and temperature. On a first approach, you would set up the crystallization experiments at three different temperatures: 8ºC, 12°C, and room temperature. There are of course many more variations on this theme to try on - please enquire with the MSKC staff on how best start crystallizing your protein.
Question: How pure does my protein have to be?
Answer: As a rule of thumb, a major band on a Comassie stained SDS-PAGE gel is often sufficient. Remember that pioneering biochemists routinely purified proteins from crude extracts by recrystallization! As with all rules, they are made to be broken and some complex proteins and enzymes will need extensive purification to produce diffraction quality crystals. Examples include kinases which often need further purification to improve homogeneity given the spread of post-translational modifications such as phosphorylation. On the same lines, think of using recombinant factories that make protein with simpler PTMs, e.g. yeast versusmammalian cell lines, and, if permitted by your studies, overexpressing globular individual domains as opposed to multiple domains linked by floppy linker sequences. Additionally, you can analyze your protein sequence for crystallizability and suitable construct design by using online tools such as XtalPred and SERp.
Question: How difficult is it to get a crystal structure?
Answer: Experience at MSKC suggests that getting crystals suitable for X-ray diffraction analysis at good resolution can take one or two days to grow crystals, and one or two days to solve and refine the structure. On the longer end it can take several months, years, or never!! A serious attempt at determining any crystal structure, from scratch, will typically require a commitment of a minimum of 6 months to one year. Perseverance is the key! Once you set up crystal trays check on them periodically, first on a weekly basis, then biweekly, and take notes! Interesting features might appear any time - precipitates, crystals, sea urchins, phase separation...Please enquire with the MSKC to assess the contents of the droplets - we have flowing access to the beamlines at SLAC.
Question: Can you help us with construct design?
Answer: Generally speaking, having an established recombinant system to overexpress your protein of interest would be your first step in any project aimed at a structural characterization. We expect users to have such a system, so that over-expression, purification, and crystallization can be carried on at users'convenience here. For nascent projects, we can help with basic construct design from plasmid selection, host selection, and tag selection, but we do not provide any facilities for recombinant DNA manipulation. Places such as EMBL Pepcore or ATUM Bio for expression vector design and production are good starting points for deciding which strategy to pursue. For oligo synthesis and sequencing on campus you can check with the PAN Facility.
Question: Which crystallization conditions to choose from for my protein?
Answer: The crystallization conditions to choose from are almost endless...so the first line of attack is to try generalist crystallization sparse-matrix designs, like JCSG+, PEG/ION, GRAS 1 & 2, PEGRx, Ammonium Sulfate, MORPHEUS, and SaltRx.
For a more aggresive research of crystallization conditions we've added the newest generations of screens to our repertoire. Among the latter we offer: the MORPHEUS II (featuring 35 small molecule ligands from the PDB, heavy metals for phasing, monosaccharides for stability, etc. and it is suitable for soluble and membrane proteins as well); MORPHEUS III (which includes a collection of drug-like small compound additives); the BCS Screen (a broad range grid screen of PEG 'smears'); the Wizard Precipitant Synergy 1 & 2 (including mechanistically divergent precipitant mixtures such as PEGs, organic solvents, additives, salts); the MIDAS screen (featuring non-traditional polymeric precipitants rarely introduced to traditional screens), and the MembFac/MemGold screens and MemAdvantage additive kit for membrane proteins and samples with limited solubility.
Finally your delving into the literature may help narrow down your search for a successful crystallizing reagent. Prior structural knowledge on your protein - the Protein Data Bank (PDB) is always your ally - may help you decide on which particular screen might be most useful for your project making more effective use of your precious sample. Further, you may want to go DIY by custom-designing a crystallization screen. We love to hear from you on whatever conditions (buffers/metal ions/ligands/inhibitors/etc etc) you think might make your protein happy. In that case, you create your own design using our Scorpion robot.
Question: We got crystals! What happens next?
Answer: Crystal imaging is done by the user: visual inspection of the crystal trays on a weekly basis will help determine which ones are adequate for X-ray diffraction analysis. We take them to SSRL at SLAC for diffraction analysis and potential data collection on one of the excellent SMB beamlines. The MSKC has streamlined access to beamline 7-1 (and other macromolecular crystallography beamlines as needed). To start this process rolling you will need to apply for access to SLAC as an MSKC collaborator. Please see the FAQ: "Question: How do I get access to SLAC/SSRL to screen my crystals as an MSKC collaborator?".
The crystal harvesting and screening process is pretty simple:
- First, we will harvest the crystals on-site at the beamline or cryo-cool them in liquid nitrogen at the MSKC (for loading using the SSRL Automated Mounting System (SAM)).
- Second, we will collect and process the data at SLAC. The SMB FAQ can answer many of your questions regarding data collection and processing.
- Finally, we will determine the atomic coordinates of the structure, inspect electron density maps, and refine the model.
The MSKC can help you with all aspects of this process from user access, beamtime application and crystal harvesting, all the way through to structure determination.
Question: How do I get access to SLAC/SSRL to screen my crystals as an MSKC collaborator?
The MSKC has streamlined access to SSRL Beamline 7-1 that users are encouraged to utilize. You will need to register as a SLAC/SSRL user and be fully badged before you can visit SLAC/SSRL installations.
To gain access to BL7-1 for crystal screening please carefully follow the instructions below:
- Register in the SLAC User Portal:
- Click "Become a new user".
- "Which facility or facilities do you plan to use?": check "SSRL".
- "Indicate the purpose of your registration": check "collaborate on a proposal".
- "Describe the types of access desired": check "onsite and remote".
- "List your Proposal(s)": enter "Access to BL7-1 with Daniel Fernandez/ChEM-H-MSKC as part of proposal 9A72"
- "Provide Name of Proposal PI or Spokesperson": enter "Daniel Fernandez".
- "Provide Primary Beam Line / Instrument": choose "7-1, PX Restricted Range MAD & Mono".
- "Provide Scheduled Experiment Date or Anticipated Visit Date": choose a date a couple of weeks time from the date you're registering.
- Email Lisa Dunn when your registration is complete requesting "Training activation and access to BL7-1 with Daniel Fernandez/ChEM-H-MSKC as part of proposal 9A72" so that your training schedule can be activated and your badging process can be completed. Your application for User access will not be granted without this step.
- Complete the necessary training as instructed in the email that you will receive.
- Visit the SLAC badging office in person (Building 53) to get your photo taken and issued with your SLAC user badge:
Question: We would like to produce a protein at MSKC. What do we need to do?
Step 1 - Make your expression construct: This step is typically beyond the scope of MSKC activities, but we are more than happy to help you with your construct design. As a first approach, we recommend using bacteria as the host using inducible vectors with the T7 promoter. Of course, the choice of host and cloning vector depends on many factors: does your protein undergo PTMs? Is glycosylation important for function/activity? Which tag and at which position in my sequence for downstream processing? Online resources that can help you in deciding the cloning strategy for your gene of interest include the protein expression core facility at EMBL or vendors like ATUM Bio. For a simple bacterial protein expression, the following steps will be:
Step 2 - Transform your expression construct into E. coli: The easiest way is to transform chemically competent BL21 (DE3) cells with your expression construct from Step 1. We currently recommend Competent TurboCells BL21 (Genlantis) which come with a simple three-minute transformation protocol.
Step 3 - Express your protein: An example bacterial expression protocol is provided to get you up and running fast. This will likely need modifications, as you go along, to optimize the procedure for your protein.
Step 4 - Purify your protein: Simple His-tag affinity purification is often sufficient for the production of crystallization quality protein. For simple proteins we often recommend setting up crystallization trials without further purification. However, some proteins will require further purification and we have a GE AKTA Pure FPLC system available for this purpose.
Step 5 - Confirm your protein identity: An SDS-PAGE electrophoresis run is the routine check for protein expression. If needed, further bioanalytical tests include intact mass-spec or tryptic mass-spec and N-terminal sequencing. Stanford core facilities such as PAN and SUMS are available to help you with this analysis.
Question: We would like to produce a protein at MSKC...and we are a commercial user
Materials and equipment Stanford users will be billed for: briefly, the major expense you will incur for a typical protein prep help the MSKC to cover costs from equipment service contracts, equipment-intensive consumables, and replacement parts. This is billed to your PTA. For your expression and purification runs you will be expected to provide your own high-value consumables including cell strains, media, antibiotics, inducer agents, proteases. ~$900 of consumables will be sufficient for the testing of ~20 expression constructs and/or ~20 liters worth of protein expression runs. All supplies are available at Stanford discounted rates in SmartMart.
Materials and equipment Commercial users will be billed for: In addition to usage fees, commercial users will also incur indirect costs (IDC) at a rate of 57%. IDC's allow Stanford University to recover certain expenses not covered through user fees. These rates are subject to change and minor incidental costs may also be incured.
Full details can be found on the MSKC rate sheet.
Question: Do you have a cell-free protein expression system?
Answer: Yes! the Bioneer ExiProgen is a fully-automated cell-free protein expression and purification system based on E. coli machinery: simply add plasmid DNA (or linear PCR product) to the reagent block and press "GO"! The only requirement is a T7 promoter and a His-tagged protein construct; the target proteins are purified using Ni-NTA magnetic beads. Typical yields of up to 100 µg of protein may be obtained, with a purity of greater than 90%. A typical fully-automated protein expression and purification run will take approximately 6-24 hrs (dependent on reagent block used). More details are available on the Bioneer Exiprogen MSKC equipment page and Bioneer's website. Exciting uses of the system include the expression of the MscL-G22S membrane protein for reconstitution into proteoliposomes.
Question: How much will it cost to crystallize my protein?
Answer: (NOTE: The values used in this FAQ answer are based on older MSKC rates, for full details please refer to the MSKC rate sheet.) Crystallization screening of a typical protein will cost somewhere between $2k-$5k per protein.
- This estimate assumes a previously non-crystallized protein. It also assumes your protein gives hits from the screening step, and the crystals need improvements via seeding/fine screening/customized screening.
- Screening 600 conditions (6 x 96-well plates), at one given temperature, and one protein concentration (10 mg/ml), is a typical starting point.
- In general, we recommend the following 12-plate extended initial coarse screening strategy (Table below). We typically screen four plates (JCSG+, MORPHEUS, Hampton Research PEG/Ion Screen, GRAS, or other wide-range, or even, custom-made screens (or Qiagen Procomplex Suite if you are looking at a protein:protein complex) at three temperatures (4°C, 13°C and room temperature). Other configurations are possible as well depending on what you know about your protein.
- Initial coarse screening can be followed by using your top ten hits with: Fine Screen crystal optimization, MMS crystal seeding optimization, and Additive Screening.
- Ball-park cost for this strategy are detailed in the table below. Everything beyond the first line is a little guess work, as we don’t know how easily your protein will crystallize, or how much optimization will need to be performed.
- Some proteins may crystallize in the first round of screening, and no further optimization is required. Other proteins may need many rounds of optimization to produce diffraction quality crystals.
- A new mutant would likely need similar re-screening. Even a single point mutation will often change the crystallization conditions.
- There is no cost for Stanford investigators to access the SSRL synchrotron macromolecular crystallography beamlines. The MSKC staff will guide first-time SSRL users to handle and diffract protein crystals.
|Step||Screen||Temperature||Strategy||Instrument||Protein Requirement (milligrams)||Number of Units||Cost per Unit||Total|
1) Initial coarse screening
|JCSG+||X||X||X||400 conditions at 4°C, 13°C and RT (12 plates)||Douglas Oryx8||2||12||$81||$972|
|2) Fine screen crystal optimization||Top 10 Hits||X||X||X||Optimization of the top ten crystal hits (10 plates)||Art Robbins Scorpion||1.5||10||$85||$850|
|3) MMS crystal seeding optimization||Top 10 Hits||X||X||X||Optimization of the top ten crystal hits by MMS seeding (10 plates)||Douglas Oryx8||1.5||10||$92||$920|
|4) Additive screening||Top 10 Hits||X||X||X||Silver Bullets and Hampton Additive Screen (10 plates)||Douglas Oryx8||1.5||10||$103||$1,030|
|Crystal Harvesting||N/A||10 new harvesting pins and loops||N/A||10||$34||$340|
|Liquid Nitrogen and cryoprotection||N/A||10L||N/A||10||$29||$290|
|Equipment training time||N/A||~5hr||N/A||5||$75||$750|