FAQs

Basics

Who can apply?

PNCC resources are open to everyone in the global scientific community who wants to learn CryoEM. Access is given out based on peer reviewed proposals. Please contact our user office with submission questions.

What does it cost?

No cost for services and training.

Is my project ready for CryoEM?

PNCC recommends reading this review by Liz Kellog and Michael Cianfrocco. Additionally, check out CryoEM 101 by Peter Shen and Janet Iwasa.

Proposal Submission

What proposal should I apply to?

This depends on your scientific objectives. Limited access receives 8-24 hours of instrument time, focused for preliminary data. General access receives up to twenty days per year with up to two years of access, allocated in five day increments. This is for long-term significant impact research and cross-training opportunities. More information can be found on Proposal Templates themselves.

What are the proposal deadlines?

All proposals are sent for review monthly and are due by 11:59 p.m. on the 1st of the month. 

Should I include preliminary data?

Yes!  Please include any preliminary data that can help reviewers establish project feasibility and staging.  Suggestions include: SDS-PAGE gels, SEC traces, SEC-MALS, DLS, negative stain TEM or cryo-TEM images. If your proposal is missing preliminary data it may receive a limited allocation of hours restricted to sample optimization and screening. If you don't have preliminary data, apply with our Limited Access proposal. We provide screening services at PNCC to get high quality preliminary data.

How many proposals can I have?

There is no limit to the number of General Access proposals a Principle investigator may have as long as the science focus is distinct and non-overlapping. A principle investigator may only have one active Limited Access proposal at a time.

What biosafety levels are accepted?

PNCC can only work with samples that are categorized biohazard safety level (BSL) 1 and 2. If the sample can be infectious or toxic in humans, animals or plants (viruses, bacteria, toxins etc.), then the risk group (RG) must be established, licenses for shipping obtained, and safe operating procedures defined according to biohazard safety level. This includes samples containing any agent (DNA) that is obtained and derived from sources that could potentially harbor (latently) such agents (e.g. serum-derived or obtained from human cell lines). Additional guidance on risk groups can be found in Section II of the Center for Disease Control and prevention in Biosafety in Microbiological and Biomedical Laboratories 5th Edition and references therein.  The starting point for our review will be the approval from the Institutional Biosafety Committee (IBC) from the applicant's home institution and the protocols on which the approval is based. Please attach both to this proposal after the biosketches. If the final sample might be considered RG1 but preparative work involved steps at BSL2, include a copy of your home institution IBC approval indicating a down-grading of BSL during the purification to support that designation. Generally, center staff will oversee approval of RG1 agents, while acceptance of RG2 agents from US institutions will require involvement of Oregon Health Science Univeristy's biosafety officer/ IBC. This may impact project award timeframes. Shipping agents from outside the US often requires additional approval from the Center for Disease Control and/or US Department of Agriculture, and will be considered on an individual basis. We cannot receive BSL2 samples in liquid form at this time. Only pre-frozen BSL2 samples with IBC documentation can be handled.

How do I edit my proposal?

Please email our user office with any questions regarding editing proposals, including adding users.

When will my proposal be approved?

It takes approximately forty-five days for a proposal to be approved at this time. If it has been longer than this, please email our user office.

How do I see my proposal score and review comments?

In the EMSL User Portal, select proposal, on new page select “Peer Reviews.”

When does my time expire?

Limited Access expires three months after approval. General Access can last for up to two years, but each 120 hour allocation expires in six months if not used.

How do I apply for more hours?

If you applied with a Limited Use (or our previous proposal types: Standard or Rapid), then you will need to apply with a new proposal. If you have a General Access (or our previous proposal type: Programmatic), then you will need to submit an General Access Extension. With a General Access proposal, you can pre-apply for more time after you have used 60% of your allocated hours (72 hours) by submitting an General Access Extension early.

Can I add more samples?

You can only add more samples to a General Access proposal during an General Access Extension. During this time, you can add up to five samples, which are restricted to samples that align directly to the original proposal and builds upon the prior samples. All samples will be checked by our co-director, Claudia López. Any samples that are found to be outside the scope of the proposal will not be approved to be shipped on-site. 

 Scheduling

How do I schedule microscope or training access once my project is awarded?

You must have a PNCC Use and Access Agreement on-file (and an Unescorted Visitor Information Form for on-site visits). See step 2 on the Access webpage for more details. Once your paperwork is completed, email scheduling to schedule the session.

How many people from our proposal can we send for an on-site visit?

There is a limit of two users visiting on-site. Contact our user office.

Can I have multiple reservations?

Yes, but please be aware of our cancellation policy in our scheduling policies .

What is the minimum and maximum time to schedule?

Each awarded proposals can only book 5 days at a time and a minimum of 4 hours.

How do I get Krios time?

Samples must be pre-screened before requests for Krios time are accepted. This means that we must either receive images of samples analyzed under cryo-conditions or be given a strong argument for biochemical mastery of the sample. If your samples are found to be of low quality, then your reservation time will be forfeit.

Where do I send pre-frozen grids?

Ship your grids any day after Monday, but please make sure that we can receive them by Friday. Please include in the dry shipper a FedEx label to return the dewar and a copy of your grid submission form. Once you have the FedEx tracking number for this shipment please forward this by email so we can track it. As soon as we receive the shipment we will let you know. Please send the dry shipper to:

    Att: Claudia López
    Oregon Health & Science University
    LVMS Mail Code: CL-P2M
    2730 S Moody Ave
    Portland OR 97201

How many grids can I send?

Per our  scheduling policies , we request that only 11 grids are shipped to be screened in an 8 hour screening session. Up to 4 grids can be loaded for a scheduled data collection reservation. 

Will you clip my grids?

All proposals have up to four hours of scientific point of contact time for sample preparation (should be used in the same day). After the allocated four hours, users should send frozen grids or come on-site to receive training to prepare their own samples. Please be aware that if your scientific point of contact needs to clip grids; your session might be delayed. If your group does not have access to a clipping station at your institution, we can provide the information needed to purchase one from ThemoScientific. Please contact support for more information. 

Can I bring my samples on-site to be vitrified at PNCC?

Yes, only if the samples have been included in the proposal's sample table and are biohazard safety level-1. We currently cannot accept liquid biohazard safety level-2 samples. Users will have access to a Vitrobot and Leica GP2 for sample preparation. All proposals have up to four of scientific point of contact time for sample preparation or training on the equipment.

Can I send other samples than what is listed in my proposal?

This depends. All requested samples must be in the same scientific scope to be approved. During the General Access Extension, approved users can request additional samples be added to their proposal. If BSL-2 samples are added, we will require a copy of your Institutional IBC for approval. Please contact support in all other cases.

Where can I leave feedback?

You can submit a Microscope Session survey. At the end of your proposal, we will send a National Institutes of Health required End of the Project survey for you to fill out. You can also contact us with specific feedback.

Get Data

How do I get my data?

PNCC processes data and distributes it to users via the EMSL Computing facility (https://www.emsl.pnl.gov) at Pacific Northwest National Laboratory in Richland, Washington (PNNL). The EMSL computing facility features parallel HPC systems, petabytes of data storage, and dedicated high-speed data transfer capability. We offer three routes for you to access your PNCC data.

  1. The fastest transfer option is using Globus. This is the preferred method because you can get near real-time access to the data as it streams off the microscope and detector. Globus also provides a single point of access for you to see old and new data using a single endpoint rather than needing separate download links for each dataset. However, not all institutions permit Globus transfers inbound. PNCC now requires each proposal to list the Globus ID for at least one team member at time of proposal submission. Once your project is approved the PNCC data transfer team will reach out to the principle investigator and team to do a test transfer via a shared endpoint to confirm if GLOBUS works for you and to establish expected transfer speeds to your local storage. Setting up Globus access is pretty straightforward and is completely free for academics and non-profits. To get the ID go to https://www.globusid.org/create where you can register for a Globus basic account if your institution doesn’t already have an institutional license. 
  2. The second fastest transfer option to download PNCC data is to use the ARIA2 parallel download manager. For every dataset collected an autogenerated email will be sent to members of the team to notify that data is available for download. However, this option is only available after a microscope session is complete and the full dataset has been uploaded to the archive. Installation of ARIA2 is straightforward with options to compile from source or direct download of binaries for OS X and Windows. Once installed, the use of ARIA2 is very easy. At the bottom of each PNCC autogenerated dataset email is a link for specific use with ARIA2 which points to the particular dataset. Simply copy this link and launch it with the ARIA2 command line to initiate the transfer. Similar to Globus, if your local connection is intermittent or the download gets halted, ARIA2 will automatically resume after disruption. ARIA2 parallelized download options are particularly helpful for big files or when data transfer rates are slow between your institution and the PNCC archive.
  3. The slowest but most accessible transfer option is via the Environmental Molecular Sciences Laboratory's User Portal “Get Data” tab. To access the datasets, login to the EMSL User Portal then either migrate to the “Get Data” tab or click the MyEMSL data download link provided in the autogenerated email mentioned above. Note: The portal login gives you a token needed to see the data and the token can time out after awhile. If you have problems seeing the data, try logging in to the portal on another browser page then refreshing the data portal page. 

If you have any issues or difficulties, please contact our Data Team directly by email. None of these tools will perform well if you are distant from the Pacific Northwest United States and have not addressed the tuning issues described in "Configure and check your machines" below.

How will I know when I have data ready?

If using Globus, you will usually see your data on the our Globus Endpoint within 30 minutes of your session starting on the microscope. You will not be notified automatically about the start of the session but your SPOC may contact you if you have a pre-scheduled reservation. Whether or not you are using Globus to download your data, an auto-generated email will be sent to the project team members when the complete dataset is archived and available for download.  This occurs after the end of the session and can take extra time depending on the archival queue. The auto-generated email will have a subject line of “MyEMSL Notification - Data Uploaded for Project XXXXX” and the message will contain direct links to your data in the EMSL data portal and a metalink that may be used to download your data with ARIA2. Note: You may receive multiple copies of this is your dataset was incomplete upon archiving.

The automated pre-processed data will be separately uploaded automatically to the archive and an auto-generated email will again be sent to the project team when that data is available for download. Depending on the queue size, the pre-processed data may appear in the archive later than the raw data. However, the pre-processed data will be visible via Globus as soon as each file is created. 

Please contact our Data Team directly by email if you still don't see your dataset more than 24 hours after the end of your session. 

What is Globus? Why are we using it?

Globus is widely used in the scientific research community for sharing and transferring large quantities of data. It is designed to provide secure, reliable, high performance data access across multiple sites in a single interface. GLOBUS is used day in and day out to transfer PNCC data from OHSU to EMSL and moves multiple terabytes of data per day. 

Introductory information about GLOBUS is available here.

How do I to obtain a Globus ID?

In order to use Globus, you will need a Globus ID. The Globus ID identifies you to Globus, and allows you to access Globus endpoints (see below), including the EMSL Data Transfer Node (emsl#dtn) that hosts PNCC user data. Additional information about getting started with Globus is available here.

What are the minimum technical requirements for running Globus?

The normal Globus mode of operations is to use endpoints at either end of a data transfer. An endpoint is usually a dedicated data storage system with high performance network interfaces and disk storage, tuned for long distance data transfers.

It is also possible to set up a personal computer as a Globus endpoint by installing Globus Connect Personal software on it. Note: that this will probably not perform as well as a dedicated, tuned endpoint. If you have a local server-class Globus endpoint at your institution, it will likely be better to transfer data to that endpoint, then make a local copy down to your own machine.

What should I do if Globus doesn't work for me?

As mentioned above in the "How do I get my data" section, PNCC has 2 other options for data transfer. Please contact our Data Team directly if you have any questions about the other access mechanisms. Note, the auto-generated emails that you will receive when every dataset is available in the archive will also have specific instructions and links to the particular dataset. 

What are typical data transfer speeds?

The average data collection results in 2TB+ of data. At 1Gbps transfer speeds (ie: a common internal wired networking) it would take 6+ hours to transfer that amount of data locally from start to finish. The further your location is from our site, the slower data transfer rates are likely to be. The emsl#msc Globus endpoint available to PNCC users has been observed sending data via Globus to Arizona as fast as 275 megabytes/second. At that rate, one terabyte can be transferred in about an hour. In comparison, a poor cross-country connection has been seen to perform as badly as 250 kilobytes/second, in which case that same terabyte would take a month and a half to transfer demonstrating the need to tune network connections at the user institution.

Configure and check your machines.

In order to get good data transfer rates from PNCC, your receiving machine(s) must have:

  1. A reliable network connection that does not experience errors or dropped packets
  2. A fast-wired network interface (10 gigabit/second is recommended). Do not use wireless for these data transfers!
  3. Tuning of the operating system (Linux, Windows, or MacOS) that dedicates extra system memory to data transfer
  4. Fast disk or SSD storage (capable of at least 100 megabyte/second) that is large enough to hold your data set(s)

The farther you are from the Pacific Northwest in the United States, the more important #1, #2, and #3 are. See here for instructions on #3.

Background info: At long distances, many megabytes of data can be “in the wire” between computers that are far apart even when taking the speed of light in a cable into account. The computers at either end must be tuned to dedicate at least a “cable full” of their memory to each active data transfer, or they will drop data that will then have to be re-sent. This problem is noticeable at distances of about 500 miles, and can slow down transfers by a factor of 1,000 on cross-country connections. Almost no computer is tuned for this “out of the box,” but fortunately tuning guidelines are available here.

How do I test my data transfer rate?

To perform a data transfer rate test, please contact our Data Team directly through email with the subject line: Test Dataset.

When a new Globus Shared Endpoint is created for your project, a large file named 'testdata' containing random data will be placed in it.  You should download that file to test transfer rates to your institution.

If you want to test with known high-performance servers, or compare the speed of distant vs. local transfers, The Energy Sciences Network (ESNet) maintains a set of machines for such testing. They use the GridFTP protocol (which Globus uses) for maximum performance. See https://fasterdata.es.net/performance-testing/DTNs.

What is ARIA2?

ARIA2 is a multi-protocol data download tool that is available at https://aria2.github.io. ARIA2 has several useful features, including the ability to transfer multiple data streams simultaneously to improve performance. It can use the metalink to your data that is in the “MyEMSL Notification - Data Uploaded for Project XXXXX” email message. It can also resume an interrupted data transfer without having to restart.

What is the MyEMSL data portal?

The simplest but least performant way to download data is from the EMSL User Portal at https://eus.emsl.pnnl.gov/Portal. Log in to the portal with your EMSL-assigned email address and password. Click on the “Get Data” tab to see a list of instruments that have data available to you. You can navigate to the data set of interest, select the files and folders that you want to download, and then click on the “Queue Selected Files for Download.” The portal will then prepare a downloadable set of files for you anywhere from several minutes to hours. You may leave the page and return to it later; the portal will continue preparing your data.

Notes:

  1. A “Project Team Access” banner across the corner of the Upload pane indicates that the data set has not yet been released to the public. This means it is available only to approved members of the project and authorized EMSL staff. As an authorized member of the project, you should be able to download this data.
  2. If you do not see any instruments or data sets on the portal pages, make sure you go to https://eus.emsl.pnnl.gov/Portal and log in, then reload the pages. Also, select the appropriate date range for the data you are looking for.
  3. Once a downloadable file set (a “cart”) has been prepared you may download it with your web browser, or use a tool like wget, curl, or ARIA2.
Can we use rsync?

Unfortunately, rsync is prohibited by an institutional firewall on our end, so is not an option. ARIA2 is available as a command line based parallel download manager. See the "How do I get my data" section above for details about ARIA2 and contact the Data Team directly if you have questions with its implementation.

Can we send hard drives?

Yes, but this must be approved by scheduling before shipping hard drives to our facility. We prefer data to be transferred via one of the options listed above, but do make exceptions for projects where the institution is far from the center, has very poor transfer rates and has large datasets. 

What are my imaging parameters?

For all DATA COLLECTION sessions, a "METADATA_INFO.txt" file is automatically created within 30 minutes of the session data appearing on PNCC compute resources. This "METADATA_INFO.txt" contains all relevant parameters including Cs value, accelerating voltage, pixel size, exposure dose, etc. Keep in mind, that the pixel size value is coming from the cross-grating grid and can be a few % off, contact your SPOC before processing the data to get an accurate value (calibrated on the standard protein dataset). The metadata file can be found at the same directory level where you will find the "frames/" folder and the "relion3_preprocess/" folders containing your raw images and preprocessed data. Open the "METADATA_INFO.txt" file with any text editing program. Please contact your SPOC with any questions about the specific parameters or the Data Team directly if the file can not be found for some reason in your data directory. Note, the metadata file is not created for screening sessions. 

 Publications

What should I do when I publish?

Acknowledgements required for all research publications and presentations supported by use of PNCC:

  • For all presentations and posters, a shortened acknowledgement is acceptable. Include the exact phrase "Use of Pacific Northwest Center for Cryo-EM supported by NIH grant U24GM129547" on any acknowledgements/funding paragraph or slide.
  • For all research publications or press releases, the full acknowledgement is required. Include the exact phrase "A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research" in the appropriate acknowledgements/funding section. 

Co-Authorship of PNCC staff for publications involving data derived from the use of PNCC:

  • If PNCC staff only provided standard specimen and data services then co-authorship is not warranted. Instead, please recognize the expert assistance of center staff (credited by name) in the acknowledgements section of the publication.

Data sharing and archival of raw and initial processed datasets:

  • All imaging and other data generated by PNCC must be made public following established accepted practices for data validation and deposition to publicly accessible worldwide repositories (EMDB or PDB) prior to manuscript publication. Additionally, all raw and initial processed datasets acquired by PNCC will be archived using the MyEMSL archive. Users can access their project related archived data anytime and anywhere via the “Get Data” tab within the Environmental Molecular Sciences User Portal. Data is owned by the user during the awarded proposal period and for one-year following the end of the respective user proposal. During this window only the proposal principle investigator, their designated team and PNCC staff can access the data. However, 1-year after the end of each individual user proposal, all associated data for that specific proposal is made publicly searchable within MyEMSL and is available to the general scientific community. The archiving of PNCC data is automatic and does not remove the requirement to deposit data related to individual publications in worldwide repositories.  

Public access to publications for dissemination of information and tracking purposes by National Institute of Health and PNCC:

  • National Institute of Health requires all research papers describing research supported by the use of PNCC must be available to the public through PubMed Central. Additionally, NIH requires that PNCC have a copy of all resulting publications and information on all deposited structures/maps. All users of PNCC are therefore required to report all journal articles, conference proceedings, books or book chapters, contributed news articles, et cetera that include data generated by use of PNCC. Authors must upload these documents to the Environmental Molecular Sciences User Portal and link it to their awarded project within two-weeks of publication.