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HT-SAXS Mail-In Instructions the basics

Introduction    Registration    Samples    Spreadsheet    Shipping    Data    Resources    Citing   

Introduction mail-in HT-SAXS at SIBYLS

HT-SAXS workflow

Below is a schematic diagram of the HT-SAXS workflow that you may take advantage of at the SIBYLS beamline 12.3.1 at the Advanced Light Source in Berkeley:


Mail-in-flow-june7

Robotics and detector

To achieve sufficient X-ray flux for informative scattering with low protein concentration and small volumes, we designed the SIBYLS beamline at the Advanced Light Source. We employ a light path generated by a super-bend magnet to provide a 1012 photons/sec flux (1 Å wavelength). The tunable incident wavelength enables rapid adjustment of the q range appropriate for the experiment without changing the sample to detector configuration (q=4π sin(θ /2)/λ where θ is the scattering angle and λ is the wavelength).

To make SIBYLS truly high-throughput, we installed a new Pilatus3 2M pixel array detector from Dectris, which gives much faster readout times over our previous MAR165 area detector.


Tecan_picture

SIBYLS then installed a new Tecan Freedom Evo liquid handling robot that divides the time it takes to collect a full plate in half, from approximately 5 hours to 2.5 hours per 96-well plate (with the potential to collect a plate in 10 minutes over the coming year).

We believe this will be a game changer for many reasons. We will be able to accommodate more users per week, enable new screening methods, and have more time to offer SEC-coupled SAXS and time resolved SAXS.

In addition to the new robotics, we have improved instruments for higher data quality. Our new monitor of transmitted beam intensity should greatly reduce buffer subtraction errors. We have also replaced the windows for lower background. In completing these upgrades, the stage is now set for increased sample to detector distance providing access to lower q (an upgrade that will occur sometime during the summer).


Registration request beamtime in 4 easy steps

1. Request mail-in account

All SIBYLS mail-in SAXS users must first register for a mail-in account:

This action is performed on this website. If you have never registered or are not logged in there will be a request link in the upper right hand corner of the page. You may also click the button below to get started.

Request Mail-in Account

2. Register with ALS

All users must also register with the ALS User Office:

You will not have access to your data until you are registered.

Register with ALS

3. Submit RAPIDD Proposal

Users must also submit one ALS RAPIDD proposal for every three mail-in uses:

Make sure you submit a "RAPIDD" proposal and not a "General User" proposal. RAPPID proposals are for record keeping only and will not affect your chances of using our service. So far, the high-throughput capabilities of our endstation have allowed us to accept every user that has requested mail-in SAXS time.

RAPIDD Proposals

4. Book mail-in slot

After requesting a mail-in account, registering with the ALS User Office and submitting a RAPIDD proposal, you may book a mail-in slot for beamtime:

If you are logged in to this site there will be a link to book mail-in time in the upper right hand corner.

In addition, the button below will lead you to the login or the mail-in booking page depending on your login status.

Login

Samples information on plate and sample requirements

Prepare 96-well plate

Order-plate-example

Our sampling robot uses specific 96-well Full-skirt plates. You must use this particular plate for your samples to be collected. Here is a link to order plates from Fisher Scientific: Order Plates


Order-cover-example

You will also need to use the AxyMat Sealing Mat for 96-well PCR Microplates to minimize evaporation. Here is a link to order mats: Order Mats


Sample requirements

Fill plate wells with 30µL of sample and buffer. Please keep the volume consistent throughout the plate.

Three sample concentrations between 1-10 mg/ml.

For redundancy, it is recommended to collect two (identical) buffers for each concentration series, one before and one after.

Do not use greater than 2% glycerol. It has been shown that 2% provides the maximum benefit. With glycerol, the viscosity of the solution increases and bubbles can become a problem. See our Methods paper for more information.




Spreadsheet sample entry and template

Upload spreadsheet

Our robot uses a spreadsheet to determine how to run your samples. Please follow the next four steps to stage your samples for data collection:

1. Download the spreadsheet template by clicking the button below:


Download Template

2. Fill it out as follows:

  • Column A

    ContainerID. "LabName". Write the name of your lab name here (no spaces).

  • Column B

    ContainerType. "plate". Do not change this column.

  • Column C

    Well. Indicates well location. Do not change any value in this column.

  • Column D

    SampleID. Write the name for the data output file (Sample ID). Only letters, numbers and underscores are accepted (no spaces).

  • Column E

    Collect. When checked ("x"), the sample will be collected (in order, starting from the top). If there is no sample or buffer in the well, leave it blank.

  • Column F

    Directory. Each sample will have it's own directory named after the well number. For example "A1", "A2", etc. If there is buffer in the well, add a "b" after the well number. This is very important for accurate data processing.

  • Column G

    WashAfter. Indicates a wash step. When checked, the sample cell will be thoroughly rinsed three times after that well is exposed. It is recommended to wash after a concentration series.

A B C D E F G
ContainerID ContainerType Well SampleID Collect Directory WashAfter
LabName plate A1 buffer1 x A1b
LabName plate A2 protein_1_low x A2
LabName plate A3 protein_1_medium x A3
LabName plate A4 protein_1_high x A4 x
LabName plate A5 buffer1 x A5b
LabName plate A6 buffer2 x A6b
LabName plate A7 protein_2_low x A7
LabName plate A8 protein_2_medium x A8
LabName plate A9 protein_2_high x A9 x
LabName plate A10 buffer2 x A10b
LabName plate A11 buffer3 x A11b
LabName plate A12 protein_3_low x A12
LabName plate B1 protein_3_medium x B1
LabName plate B2 protein_3_high x B2 x
LabName plate B3 buffer3 x B3b
LabName plate B4 B4 (no sample)

3. Upload your updated spreadheet to the SIBYLS SAXS database

Upload Spreadsheet

4. Once you have booked a mail-in slot, a unique barcode will be created for your plate. Print this label out and tape it to the side of your plate.

Example Label:

Barcode_image_example

Shipping how to package and ship your plates

Ship plate overnight

    How to Safely Pack Your Plate:
  1. Seal plate securely with recommended sealing mat. DO NOT use adhesive based sealing film. Be sure label is securely taped to side of plate.
  2. Place sealed plate in plastic bag or wrap with parafilm.
  3. Using two 4°C cold packs (NOT frozen), place one on top and one on bottom of plate.
  4. Rubberband the assembly together for placement within styrofoam shipping box.
  5. Tighty pack styrofoam box by surrounding assembly with more cold packs.
  6. Okay if these are frozen, but do not allow plate to touch any frozen packs. DO NOT ship with wet ice.
  7. Clearly label the outside of your shipping box with the temperature of your samples. This is extremely important if a shipping delay occurs.
  8. Provide us with a tracking number

Shipping address

Ship samples overnight to arrive at SIBYLS on the first day of your shift:

    SHIPPING ADDRESS:

  • Lawrence Berkeley Lab
  • ATTN: Kathryn Burnett / SYBILS 12.3.1
  • 1 Cyclotron Road MS 6R2136
  • Berkeley, CA 94720
  • Phone: 510-486-5378

Data what happens when and after your plate arrives

SIBYLS staff collect data

    The following outlines how your samples will be processed:
  • You will be notified by email that your samples have arrived.
  • The plate will be stored at the appropriate temperature: 4°C or -80°C.
  • It will be lightly spun at 3700 rpm for 10 minutes at 4°C prior to data collection.
  • It will be held at 10°C during data collection, which lasts approximately 2.5 hours for a full plate.
  • You will be notified by email as soon as your data is available.

SIBYLS staff process data

You will get back integrated scattering profiles in ASCI format in a results directory for each sample. A series of exposures, in equal sub-second time slices, are taken of each well.

In addition, you will receive a report with an initial assessment of the sample from our beamline staff.

Accessing your data

You will receive an email once the data package is available. The results will be stored and accessible in your data directory i.e. /data/username/ on our "kona" or "bl1231" servers. Where username is your individual or lab's login name.

To access data in your remote SIBYLS directory open a Terminal window and log into a server (example shown for "bl1231", substitute bl1231 with kona to use "kona"):

local>ssh username@bl1231.als.lbl.gov

Then once logged in change directories to your data directory and list the contents:

[username@bl1231 ~]$ cd /data/username
[username@bl1231 ~]$ ls

To transfer files to your local computer, open a Terminal window and make or navigate to target directory to deposit your results or data. Example:

local> mkdir saxs
local> cd saxs

Then transfer the files using either remote sync rsync or secure copy scp, for example:

local > rsync -avz username@bl1231.als.lbl.gov:/data/username/20180101_username_Results .

would copy over the entire 20180101_username_Results directory from our server to your local machine using rsync and the "bl1231" server.

An example of just copying over your .dat files using scp and the "kona" server would be:

local>scp -rp username@kona.als.lbl.gov:/data/username/20180101_username_Results/*.dat .

Type man rsync or man scp in your Terminal for more information.

You may also use sftp or other GUI-based tools to transfer data to your local computer.

This is helpful in particular if you want to reprocess unsubtracted data in ScÅtter. The ScÅtter tutorial will help you navigate data processing.

If you have any questions, please contact Kathryn.


Resources where to get additional information

Feedback

We are continuing to improve our systems with the aim of making data of higher quality. Please provide us with constructive feedback. For additional information please feel free to contact:

  • Kathryn Burnett
  • Research Associate
  • kburnett@lbl.gov
  • Greg Hura, Ph.D
  • SAXS Scientist
  • glhura@lbl.gov

Additional details

Learn about SAXS collection at SIBYLS...

README

Learn about common problems with SAXS samples...

SAXS Analysis

Online Webinars

We regularly present online webinars to highlight new SAXS features, and how to address and solve specific problems that occur during SAXS data collection and processing:


Citing the key to keep SIBYLS running to serve you

Cite SIBYLS in published papers

Please cite all relevant SIBYLS beamline papers below in the 'References' section of your journal article or book chapter

  • Dyer KN, Hammel M, Rambo RP, Tsutakawa SE, Rodic I, Classen S, Tainer JA, Hura GL (2014) “High-Throughput SAXS for the Characterization of Biomolecules in Solution: A Practical Approach” Methods Mol. Biol. 1091, 245-58. (Specific Methods paper for HT-SAXS at SIBYLS)
  • Classen S, et al. (2013) “Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source” J. Appl. Crystallogr. 46(Pt 1), 1-13. (SIBYLS Beamline hardware)
  • Hura GL, et al. (2009) "Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)" Nature Methods 6, 606–612. (General High Throughput SAXS)
  • Putnam CD1, Hammel M, Hura GL, Tainer JA (2007) "Solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution” Q. Rev. Biophys. 40, 191-285. (General SAXS)

Acknowledge SIBYLS

Please include the following in the 'Acknowledgements' section of your journal article or book chapter when using data generated at SIBYLS:

"This work was conducted at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10OD018483."

These citations and acknowledgments are absolutely essential in keeping the mail-in SAXS program funded and the beamline continually repaired and upgraded, as well as to provide access to experienced staff and the development of new technologies, such as SEC-SAXS.