1

Cryo-EM Core Facility (CEMF) - Annual Report 2019

Table of Content: page

1) Progress Report and News 2019 – Summary ………………………………………………………………… 1

2) Statistics about the CEMF usage …………...………………………………………………………………… 2

3) Full annual report CPRIT Core Facility Award ...………………………………….………………………… 4

4) Considerations for data collection strategies (multi-hole/shot) – [recommended read for certified users] …. 14

1) Progress Report and News 2019 – Summary:

Purchases: a second Titan Krios G3i with Bioquantum energy filter, K3 direct electron detector, Fringe-

free illumination upgrade (UTSW funded), Glacios with MicroED capability (CPRIT funded), Falcon 3

direct electron detector (HHMI funded) => ETA 6/2020, expected installation on South campus: ~3 months.

South campus EM suite (L building): 95% architectural plans done, renovation completion before 6/2020.

New CEMF staff members: Jose Martinez-Diaz to assist the facility manager Dr. Daniel Stoddard with the

maintenance and daily operation of the electron microscope cryo-EM suite(s), and Eric Zhang to upgrade

and maintain the facility IT infrastructure and handle other IT aspects.

Increased throughput for single particle cryoEM data collection: several factors have contributed to this

increase, such as the K2-to-K3 upgrades (Talos: 10/2018; Krios: 3/2019) and implementation of multi-shot/

-hole strategies using (fast) image shift; this allows collection of ~5000-8000 images/24 hours on the Krios.

On-the-fly processing: allows users to receive motion-corrected images automatically within ~10 min. after

collection on the microscopes, allowing the users to adjust their data collection in real-time. A “user

manual” with details was email to our cryoEM mailing list and was posted on our website under “News”

(www.utsouthwestern.edu/labs/cemf/assets/OnTheFly-Processing-Manual-UTSW-CEMF-DN.pdf). After

visiting the UCSF cryo-EM facility, we set-up a custom-built solution at UTSW in collaboration with the

UTSW BioHPC Facility (Dr. Murat Atis), i.e. we purchased a CEMF-dedicated, high-end GPU workstation

with 8 TESLA T12 cards housed in the BioHPC data hall, and implemented scripts that automatically

perform motion correction of the raw movie data before distributing both the raw and processed data to the

users (CEMF-dedicated BioHPC storage space). The system is still being tested (and 2 GPU cards need to be

exchanged under warranty), but once fully established we plan to further expand functionality of the pipeline.

Communication - new: we will post a monthly facility manager progress reports from Dr. Daniel Stoddard

at the top of the “News” section of the CEMF website (https://www.utsouthwestern.edu/labs/cemf/news/) to

update users about progress and ongoing projects in the CEMF; this new service will start 12/01/2019.

New email address for the Internal Advisory Committee (IAC): [iac2cemf@utsouthwestern.edu] will

send your comments or suggestions directly to all standing members of the IAC of the CEMF.

Quality control: the performance of the CEMF instruments is checked on a regular basis by the CEMF

staff; from now on, we will note the results of the latest resolution limit tests in the instrument log books

[see e.g. 11/7/2019 for Krios: full visible ring at 1.17Å, and 11/8/2019 for Talos: full visible ring at 1.44Å].

Conference visit: Dr. Stoddard (facility manager) attended the GRC for 3D EM in Hong Kong June 2019.

Other statistics for the past 12 months:

30 labs from 15 different departments/centers used the CEMF;

14 new CEMF users were trained (2 user training courses offered in parallel with 1 day/week on each TEM);

21 papers acknowledging the CEMF were published (involving 9 different CEMF user labs);

34 Krios-time applications from 11 different labs were approved.

Remote monitoring: this is in progress to establish remote monitoring of data collection for users.

2

2) Statistics about the CEMF usage in the past 12 months

Please note that user fees are per hours and identical for all users/labs.

2.1. Total microscope hours (Krios + Talos) by groups:

Nicastro – Bai – Li – Hibbs –SBL- Erzberg.–ZO - Jiang– admin–Rosenb.–Orth– Yu - Rizo - Zhang- others

2.2. Talos total hours used (note: K3 install 10&11/2018, summer 2019 repair due to lost grid & vendor delays)

downtime

3

Talos use by groups: (note: user fees are per hours and identical for all users)

SBL– Li – Hibbs- ZO – Erzb.–Jiang–Bai- admin- Rosenb.-Rizo-Yu– Orth- Beutler-Wang-Zhang-others

2.3. Krios total hours used: (note K3 installation 3/2019)

4

Krios use by groups:

Nicastro – Bai – Li – Hibbs – Erzberger –Jiang– Rosenb.–admin– Orth – Zhang – SBL – others

3) CPRIT Core Award – Full Annual Report 2019

Annual Progress Report Grant Year Ending 30 Aug 2019

Grant ID: RP170644

Title: Establish a new Cryo-Electron Microscopy Core Facility and service for structure determination at UT Southwestern Medical Center

PI/PD/CR: Daniela Nicastro

Organization: The University of Texas Southwestern Medical Center

Key Accomplishments

1. Purchase of a second cryo-electron microscope (Glacios) to increase sample screening throughput.

2. Implementation of IT infrastructure improvements, “on-the-fly” image processing, and faster data acquisition

strategies (called “multi-shot” and “multi-hole”).

3. Hired and trained a new Cryo-EM Core Facility staff member.

4. The Structural Biology Lab (SBL) has provided single-particle cryo-EM services, including training and

experiments, to 21 UT Southwestern research laboratories in 2019. A SBL staff member has attended a cryo-

EM workshop at the Pacific Northwest Center for Cryo-EM (PNCC) for further training and is disseminating

this knowledge to the cryo-EM research community at UT Southwestern.

5. 33 cancer-related projects have been pursued in the Cryo-EM Core Facility by 20 UT Southwestern research

groups. 20 published and 3 submitted papers acknowledge CPRIT support of the Cryo-EM Core Facility.

5

Summary of Goals and Objectives

Goal 1 – Objective 1. Equipment purchases

Summary of Progress: 1. We have completed the purchase order for a Glacios 200 kV cryo-electron

microscope that will increase the throughput of cryo-sample screening in the future. Delivery of the

instrument is planned for 6/2020.

2.The Glacios TEM was purchased with “microcrystal electron diffraction” (MicroED) capability (i.e. with

special MicroED CEMOS detector and beam stopper). This method combines cryo-EM with electron

diffraction and crystallography data analysis to solve crystal structures of inorganic nanomaterials, small

molecules and proteins at high resolution, while using nanosized crystals that would be too small for x-ray

crystallography. Thus, MicroED has important applications in new drug design and discovery in

nanotechnology and cancer therapy.

3. Initiated by Dr. Rosen, the UTSW HHMI investigators have successfully acquired HHMI funding to

purchase a Falcon 3 direct electron detector (Thermo Fisher Scientific) for the Glacios TEM, which will allow

data collection for medium resolution structures (similar to our Talos Arctica with K3 direct electron detector)

and methods development in the future.

4. Renovation South campus cryo-EM suite: The Glacios and a second Titan Krios G3i (for details see below)

will be housed in a newly renovated, climate-controlled cryo-EM suite on South campus (L1.236, about 10-15

min. from the existing North campus cryo-EM suite). We have just received the “95% architectural plans” and

construction will begin 12/2019 to ensure completion before 6/2020.

5. We have upgraded our two Gatan K2 direct electron detectors (North campus Talos Arctica and Titan Krios

G2) to Gatan K3 direct electron detectors. The K3 cameras have a larger imaging area and faster frame read-

out, which has increased the speed of data collection by a factor of ~4.

6. Faster data collection strategies: We have implemented “multi-shot and multi-hole” data collection strategies

on the Titan Krios and “multi-hole” on the Talos Arctica; both strategies use image shift (rather than slower

stage shift) to record multiple images from samples areas in close proximity. Together, these upgrades have

increased our data acquisition rate on the Titan Krios G2 to 5000-6000 images/24 hrs, compared to previously

~1000 images/24 hrs.

7. The purchase of a sample dispersion robot (Objective 1 iii) has been postponed to year 3 because the

commercial version of the sample dispersion robot Spotiton (now named Chameleon) is still under

development at the company TTP LabTech.

Anticipated Activities for Year Ahead: 1. Renovations and instrument installation: We will complete the

renovation of the South campus cryo-EM suite (funded by UT Southwestern) and install the new Glacios and

Titan Krios G3i microscopes starting June 2020. We anticipate that the South campus expansion will be fully

operational Q4 2020.

2. Software upgrades: We will receive two software upgrades that will further increase ease and throughput of

cryo-EM data collection: A) Installation of the Thermo Fisher Scientific “Advanced Scripting software” is

planned for Nov. 2019. This upgrade should allow for implementation of automation scripts, e.g. for

expanded multi-shot/multi-hole data collection strategies, and for automated operation of the Volta-Phase-

Plate. The latter accessory increases the image contrast and is routinely used for cryo-electron tomography

data collection; however, currently it requires manual adjustments by the users many times during a 24-hour

data collection session. B) The Thermo Fisher Scientific EPU software was the preferred single particle cryo-

EM data acquisition software on the Titan Krios G2 with K2, but currently it is not compatible with the K3

camera. We anticipate installation of an upgraded EPU software with K3-compatibility on the Titan Krios

G2 in Q1 2020. The company has not yet confirmed if the upgraded EPU software will also be compatible

with the “non-embedded” K3 on the Talos Arctica.

3. Sample dispersion robot: We will test available sample dispersion robots (in addition to TTP Labtech’s

Chameleon, other robots are under development); the prices will likely be higher than the budgeted estimate,

therefore we will use the test data of the most suitable instrument for our user base as preliminary data to

apply for additional funding (shared instrumentation grant, 5/2020), and then purchase a sample dispersion

6

robot for the SBL facility to increase for robustness, repeatability and thus throughput for cryo-sample

preparation.

Additional Comments: We have also completed the purchase order of a second Titan Krios G3i (high-end)

cryo-electron microscope (~$10 million, funded by UT Southwestern). Delivery of the instrument is also

planned for June 2020. We also plan to purchase the recently announced “Fringe Free Illumination” (in-factory)

upgrade for this newly purchased Titan Krios G3i. UT Southwestern is also funding the ~$2.5 million

renovation of the cryo-EM suite on South campus to house the two new TEMs. This is a reflection of the great

success of and demand for the Cryo-EM Core Facility, as well as the strong support by the UT Southwestern

leadership. In the SBL facility, a multi-angle light scattering (MALS) instrument has been installed in-line with

the SBL FPLC instrument to enable sample analysis at 4 degree C. This will enable users to critically evaluate

sample monodispersity prior to applying the sample to cryo-EM grids.

Goal 2 – Objective 1. Single Particle Service

Summary of Progress: The UT Southwestern Structural Biology Laboratory (SBL) that is directed by Dr.

Diana Tomchick, has continued to provide Service for Single-Particle cryo-EM (SSP) to both cryo-EM expert

and non-expert labs at UT Southwestern and to two local outside users. The SBL services include: a) providing

weekly SBL-operated screening sessions on the Talos Arctica for rapid and highly efficient sample screening,

b) providing training for use of accessory and sample preparation instruments that are housed in the SBL

facility, and c) providing "experiments", i.e. cryo-EM grid preparation, data collection, image processing and

structure determination. As can be seen from the attached list of the 30 laboratories (from 15 different

departments) that have used the UT Southwestern Cryo-EM Core Facility in 2019, 20 groups have pursued a

total of 33 cancer-related cryo-EM projects (60% of all projects), showing that there is a large emphasis on

supporting cancer-related projects in the Cryo-EM Facility. Our users have published 20 papers acknowledging

support by the CPRIT Core award; 3 additional manuscripts are submitted and several more are in preparation.

21 of the 30 labs have used the SBL for either training and/or experiments, and 8 non-expert labs (7 with

cancer-related projects) have used the full service of the SBL and list the SBL as "collaborator" (including

authorship on future publications). In August 2019, a staff member of the SBL, Dr. James Chen, attended the

three-week Summer Cryo-EM Workshop offered by the Pacific Northwest Cryo-EM Center (PNCC), which has

greatly increased his knowledge and skill level in all aspects of the single-particle workflow. Dr. Chen has

worked with Dr. Stoddard (the facility manager of the Cryo-EM Core Facility) to implement the “multi-hole”

image acquisition strategy on the Talos Arctica, and is disseminating his knowledge to the user community

through an upcoming seminar in the “structure group” discussion meeting (SBWIP).

Anticipated Activities for Year Ahead: The SBL will continue to provide training and experimental service to

UT Southwestern research labs that require cryo-EM for their studies. We will also continue to grow the

number of cancer-related projects pursued in the Cryo-EM Core Facility in year 3. To keep up with the growing

demands and user base, the SBL plans to hire a technician in Q4 2019 to assist with general lab work, including

assisting with sample preparation and plunge-freezing of cryo-EM grids. To be able to make full use of the

MicroED capability of the to-be-installed Glacios TEM and provide MicroED service in the future, 1-2 staff

members of the SBL will visit a MicroED expert lab (e.g. Tamir Gonen, UCLA) 2020 to be trained.

Goal 2 – Objective 2. Administrative plan for sustainability

Summary of Progress: The previously reported, administrative plans for (partial) cost recovery for both the

CEMF and the SSP/SBL have been followed as planned. The fees for electron microscope instrument usage

have increased in the fiscal year 2019 to $20/hour for the Talos Arctica, and $25/hour for the Titan Krios G2,

and the fees will increase in the fiscal year 2020 to $25/30 per hour (as of Sept. 2019). The CEMF facility

manager training and SSP/SBL staff service fees are $60/hour. Please see the attached supplements for more

detailed information about the SSP/SBL fee structure.

7

Anticipated Activities for Year Ahead: We will continue our outreach efforts to inform the cancer research

community at UT Southwestern as to how their research could benefit from using the Cryo-EM Core Facility

and SBL cryo-EM services. An information session led by the SBL staff is planned for Q1 2020.

Goal 3 – Objective 1. Educational programs

Summary of Progress: Dr. Xiaochen Bai (CEMF Co-Director) is teaching single particle cryo-electron

microscopy as part of the graduate school core course in the UT Southwestern Life Science graduate program.

Dr. Xuewu Zhang (user of the Cryo-EM Core Facility and chair of the graduate course “Modern Methods in

Structural Biology (MMSB)”) and Dr. Daniela Nicastro (CEMF Director) are teaching “model building and

refinement of cryo-EM structures” and “general electron microscopy methods”, respectively, in the MMBS

course as part of the Molecular Biophysics graduate program curriculum. Drs. Bai (CEMF Co-Director) and

Stoddard (CEMF manager) attended the Gordon Research Conference for Three Dimensional Electron

Microscopy June 2019 in Hong Kong, China, to learn about novel techniques and breakthroughs in 3D EM of

biological structure determination. Dr. Stoddard is part of an initiative that established a Slack channel (group

forum) for cryo-EM facility managers worldwide. SBL staff members are hosting and presenting seminars in

the Structural Biology “work-in-progress” (SBWIP) seminar, as well as a Yammer group-forum for real-time

chat and sharing of files, where UT Southwestern cryo-EM researchers can post questions and answers, or

search previously archived topics.

Anticipated Activities for Year Ahead: In the upcoming year, the two cryo-EM experts in the SBL facility,

Drs. James Chen and Yang Li, will be joined by Dr. Yan Han (a recent recently hired cryo-EM expert that will

provide cryo-EM service for Dr. Youxing Jiang and other HHMI investigators at UT Southwestern), and

together they will collaborate with the BioHPC Facility at UT Southwestern to implement an image processing

and analysis workshop/nanocourse in Spring 2020.

Goal 3 – Objective 2. Staff and training program

Summary of Progress: In anticipation of our facility expansion, where two additional electron microscopes

will be installed in the South campus cryo-EM suite starting 2020, we have hired two additional Cryo-EM Core

Facility staff members, Jose Martinez Diaz, to assist the facility manager Dr. Daniel Stoddard with the

maintenance and daily operation of the electron microscope cryo-EM suite(s), and Eric Zhang to upgrade and

maintain the facility IT infrastructure. Jose Martinez Diaz joined the Facility April 2019 and is now fully trained

to load cryo-samples, assist users with data collection, troubleshoot instrument errors and interact with service

engineers from the instrument manufacturers. We have continued to offering two user training courses (one on

each electron microscope) in parallel, with one day per week dedicated to user training on each microscope. Dr.

Daniel Stoddard has trained and certified 14 new “hands-on” users in the Cryo-EM Core Facility over the past

12 months.

Anticipated Activities for Year Ahead: Dr. Stoddard will continue to update the syllabus of the training

course as needed. Cryo-EM facilities at different academic institutes employ different philosophies about

“hands-on” training of users and the degree to which users operate the microscopes, from the “no user training”

model at the University of Massachusetts in Worcester, MA, where all data are collected by facility staff, to

facilities where all users are from a few EM expert labs and are trained to expert-level. We will evaluate

different options to provide e.g. a two-tier, i.e. medium- and expert-level training program, in which medium-

level users would learn to acquire data, but not to align the microscopes, instead staff would then expertly align

the scopes as fee-for-service for these users, whereas expert-users from specialized labs will be completely

independent.

8

Goal 4 – Objective 1. High pressure freezer for cryo-FIB

Summary of Progress: The Wohlwend Compact 3 high-pressure freezer has been set-up in the conventional

EM Core Facility (directed by Dr. Kate Luby-Phelps), which is located directly next to the North campus cryo-

EM suite (in NL1). The high-pressure freezer is expertly operated by a staff member of the conventional EM

Core Facility, Dr. Anza Darehshouri. So far, various samples have been successfully frozen for five UT

Southwestern labs: embryonic cardiomyocytes for Dr. Sadek’s lab, Drosophila embryos for Dr. Elizabeth

Chen’s and Dr. Doubrovinski’s lab, budding yeast for Dr. Friedman’s lab, as well as yeast, Chlamydomonas, C.

elegans worms and zebra fish embryos for Dr. Nicastro’s lab. Because of the persisting technical issues with

cryo-FIB milling of thick samples (e.g. high pressure frozen samples; for details see Goal 4), the quality of

vitrification by the Wohlwend Compact 3

high-pressure freezer could so far not be evaluated by the Nicastro lab using cryo-EM. However, the structure

preservation of all remaining samples was satisfactory as evaluated by conventional TEM of high-pressure

frozen, freeze substituted samples.

Anticipated Activities for Year Ahead: In the year ahead, a main focus will be to establish protocols that will

allow us to successfully cryo-FIB mill high-pressure frozen thick samples. Once successful, this would allow us

to evaluate if the Wohlwend Compact 3 high-pressure freezer generates truly vitreous (meaning ice-crystal free)

samples. 1-2 postdoctoral fellows from the Nicastro lab will be trained to operate the high-pressure freezer

independently, to facilitate more efficient use of this instrument.

Goal 4 – Objective 2. Cryo-FIB pipeline

Progress Against Timeline: Behind Schedule

Explanation and/or Comments: We continue our efforts to beta-test the new cryo-FIB instruments; however,

despite receiving several hardware-upgrades for our originally purchased cryo-Scios DualBeam with cryo-FIB

(beta-test instrument) and hiring a dedicated staff member, Evan Reetz, to operate the instrument daily, we have

only been able to establish routine operating procedures for milling lamella from relatively small cells (up to 10

micrometer diameter), which can be properly vitrified by plunge-freezing. Larger cells, tissues and small

organisms, however, require high-pressure freezing for vitrification, and we – like all other labs in the world -

have been struggling with technical issues to cryo-FIB mill thick high-pressure frozen samples. The likely

reason for the technical issues is "stress-buildup" in the sample due to charging and inherently poor conductivity

of cryo-samples. Indeed, based in part on our experiences and technical input, Thermo Fischer Scientific has

abandoned the cryo-Scios system, and - as written into our contract and described below (Point 2 in Summary

of Progress) - has replaced our Scios instrument with an improved Aquilos instrument. More time than

anticipated is needed to solve these technical issues. Nonetheless, we remain confident that our efforts will pave

the way towards high-resolution structural studies inside eukaryotic cells and tissues.

Summary of Progress: 1. The manuscript of our first cryo-FIB study – which we reported in the last progress

report as "under review" - has now been published (Hariri et al. JCB 2019).

2. The manufacturer of our cryo-FIB has recently (Sept. 2019) replaced our cryo-Scios with an improved

Aquilos instrument for cryo-FIB milling. Evan Reetz, Dr. Nicastro, and 3 postdoctoral fellows from the

Nicastro lab received 4 days of applications training by Thermo Fisher Scientific on our new Aquilos

instrument in early October 2019.

3. Our “diving board” milling strategy (where the lamella is severed from the bulk-sample on one side to relieve

“stress”) allowed us to successfully cryo-FIB mill a few lamellae from high-pressure frozen samples.

However, the lamellae were not stable enough in the electron beam for cryo-ET imaging in the Titan Krios.

Further experiments are underway to improve stability of "dive-board" lamellae, e.g. by re-annealing them to

the "bulk-sample after the milling.

9

Problems/Issues Encountered: Most eukaryotic cells (let alone tissues and organisms) require high-pressure freezing for vitrification to avoid ice crystal damage of the cellular structures, and methods for effective cryo-FIB milling of these thick samples are still under development. Actions Taken to Address Problems/Issues: We have received several hardware upgrades and ultimately a completely new cryo-FIB instrument (Aquilos). See also “anticipated activities” for 2020. Anticipated Activities for Year Ahead: A recent publication from the Baumeister lab (DOI: 10.1038/s41592-019-0497-5) showed that the “cryo-FIB lift-out technique” enables molecular-resolution cryo-electron tomography of C. elegans tissue. Thermo Fisher Scientific has recently announced their “easy-lift-out system”, which will become available summer 2020. We plan to test and – if successful – purchase the “easy-lift-out system” for our Aquilos (~$300k) to be able to establish successful operating procedures for thick sample cryo-FIB milling.

Goal 4 – Objective 3. Cryo-FIB pipeline projects

Progress Against Timeline: Because of the technical issues with cryo-FIB milling of thick samples (including

mammalian cells cultured on EM grids to study DNA damage responses), we have expanded the number of

projects that use relatively small cells (with a diameter of up to ~10 micrometer), such as budding yeast

(projects: septin cytoskeleton – one manuscript in preparation, phase-separated nuclear bodies, lipid droplet

biogenesis, and protein aggregation) and a structural study of the parasite Toxoplasma, which can all be vitrified

by plunge-freezing. Six cryo-FIB projects – all with cancer-relevance – were pursued in the Cryo-EM Core

Facility (see attached user and project list under Goal 2/Objective 1).

Anticipated Activities for Year Ahead: We will continue to pursue projects using cryo-FIB milling of

relatively small, plunge-frozen cells, but as soon as either the cryo-gripper or an easy-lift-out system is installed

on our Aquilos we will expand our projects to larger cells to study a wide range of cancer-related questions.

Goal 5 – Objective 1. Storage space for data

Progress Against Timeline: In the last funding period neither the Cryo-EM Core Facility, nor the SBL needed

to purchase additional data storage space on the high-performance computer cluster (BioHPC) at UT

Southwestern or for local storage drives.

Anticipated Activities for Year Ahead: We will require and purchase additional data storage space once the

two new electron microscopes have been installed on the South campus.

Goal 5 – Objective 2. Data transfer pipeline

Progress Against Timeline: We established an “on-the-fly” data processing pipeline: Beam-induced sample

motion during cryo-EM data acquisition leads to image blurring and reduces resolution of the 3D

reconstructions. However, motion-induced blurring can be minimized by recording a “movie” consisting of

hundreds of very quickly recorded images (“frames”), instead of recording a single long exposure image. The

first image processing step in cryo-EM is then to perform beam-induced motion correction by aligning the

frames before flattening each movie into one image. To automate and increase efficiency of the image

processing workflow at UT Southwestern, we have performed the following upgrades to the IT infrastructure of

the Cryo-EM Core Facility during the last funding period: A) A delegation from the UT Southwestern Cryo-EM

Facility (Drs. Xiaochen Bai, Daniel Stoddard) and the BioHPC Facility (Dr. Murat Atis) visited the cryo-EM

facility at the University of California San Francisco (hosted by Yifan Cheng) to evaluate their IT infrastructure;

B) in the UT Southwestern Cryo-EM Core Facility, we have upgraded the automated data

transfer/synchronization scripts from the local K3 camera computers (from the Titan Krios and Talos Arctica)

to the high-performance computing cluster BioHPC at UT Southwestern, C) we installed a CEMF-dedicated,

high-end GPU workstation with 8 TESLA T12 cards in the BioHPC data hall, and D) we set-up scripts that

10

automatically perform motion correction of the raw movie data before distributing both the raw and the

processed data to the users (in CEMF-dedicated BioHPC storage space). The high speed Ethernet connection,

hardware upgrades and automation scripts reduce the initial image (pre)processing step from before ~2-3 days

to now ~10 minutes, allowing users to obtain immediate feedback information for each collected movie, e.g. to

evaluate the quality of the currently collected data. The “on-the-fly” processing pipeline is designed to be user-

friendly, i.e. users have to specify only two parameters (dose rate and image pixel size) at the beginning of their

data collection session and everything else runs automatically. As an added benefit, the motion corrected and

flattened images have greatly reduced file-sizes and thus can be transferred more rapidly by the users to their

lab-specific storage devices.

Anticipated Activities for Year Ahead: In the year ahead, we plan to further improve the “on-the-fly” data

processing pipeline by adding automation of more advanced processing steps to our existing “on-the-fly”

system. These advanced features will include: a) CTF estimation of the motion corrected images, b) fully

automated particle picking through machine learning approaches, c) High speed 2D classification of auto-

picked particles using the software package Cryosparc. The class averages generated by this expanded “on-the-

fly” system will then provide even better feedback to the users about the (currently) recorded data quality. We

will implement remote operations, meaning the capability to operate the electron microscopes from remote

workstations. In phase I, this will be installed for the South campus cryo-EM suite, before expanding it in phase

II to the existing North campus cryo-EM suite.

Publications and Facility Users

See table next pages

# PI Name (Dept) Funding Sources

SSP (SBL) usage:

"training" and/or

"experiments"

UTSW collaborator Project Title Cancer RelevanceGroup 1-4

(see below)Publications

1. Shah V*, Zhao X*, Lundeen RA, Ingalls AE, Nicastro D, Morris RM (2019)

Morphological plasticity in a sulfur-oxidizing bacterium from the SUP05 clade

enhances dark carbon fixation. mBio DOI: 10.1128/mBio.00216-19. [PMCID:

PMC6509183]

2. Spietz RL, Lundeen RA, Zhao X, Nicastro D, Ingalls AE, Morris RM (2019)

Heterotrophy within a sulfur-oxidizing lineage of chemoautotrophic marine

bacteria. Environ Microbiol. DOI: https://doi.org/10.1111/1462-2920.14623

4

1. Dymek EE, Lin J*, Fu*, Porter ME, Nicastro D, Smith EF (2019) PACRG and

FAP20 form the inner junction of axonemal doublet microtubules and regulate

ciliary motility. Mol Biol Cell 30:1805-1816. [PMCID: PMC6727744]

2. Lin J*, Le TV*, Augsperger K, Tritschler D, Bower R, Fu G, Perrone C,

O’Toole ET, VanderWaal Mills K, Dymek EE, Smith EF, Nicastro D*, Porter ME*

(2019) FAP57/WDR65 targets assembly of a subset of inner arm dyneins and

connects to regulatory hubs in cilia. Mol Biol Cell mbcE19070367. doi:

10.1091/mbc.E19-07-0367 [Epub ahead of print]

3. Fu G, Zhao L, Hou Y, Dymek E, Loreng TD, Song K, Shang Z, Phan N, Smith

EF, Witman GB, Nicastro D (2019) Structural organization of the C1a-e-c

supercomplex within the ciliary central pair apparatus. (accepted J Cell Biol)

4. Gui L, Song K, Tristchler D, Bower R, Si Y, Dai A, Augsperger K, Sakizadeh J,

Grzemska M, Ni T, Porter ME, Nicastro D (2019) Scaffold subunits support

associated subunit assembly in the Chlamydomonas ciliary nexin-dynein

regulatory complex. (accepted PNAS );

5. 1 manuscript submitted.

and 5 manuscripts are in preparation;

Dawn Wetzel, MD, PhD

(Pediatrics - Infectious

Disease)

4

Hariri H, Speer N, Bowerman J, Rogers S, Fu G, Reetz E, Datta S, Feathers JR,

Ugrankar R, Nicastro D, Henne WM (2019) Mdm1 maintains endoplasmic

reticulum homeostasis by spatially regulating lipid droplet biogenesis. J Cell

Biol . jcb.201808119. [PMCID: PMC6446837]

4

4 1 manuscript in preparation

4

4

Kendra Fredrick, PhD

(Biophysics)4

Michael Reese, PhD

(Pharmacology; CPRIT

RP160157)

4

Perry Bickel, MD

(Internal Medicine -

Endocrinology)

4

Xuewu Zhang, PhD 1-3

Li J, Shang G, Chen YJ, Brautigam CA, Liou J, Zhang X, Bai XC (2019). Cryo-

EM analyses reveal the common mechanism and diversification in the activation

of RET by different ligands. eLife 8:e47650 doi: 10.7554/eLife.47650.

4

Uchikawa E, Choi E, Shang G, Yu H*, Bai XC* (2019) Activation mechanism of

the insulin receptor revealed by cryo-EM structure of the fully liganded

receptor–ligand complex. eLife 8:e48630. doi: 10.7554/eLife.48630

(*corresponding authors)

4

Li J, Choi E, Yu H*, Bai XC* (2019) Structural basis of the activation of type 1

insulin-like growth factor receptor. Nature Commun 10:4567 doi:

10.1038/s41467-019-12564-0 (*corresponding authors)

Users of the Cryo-EM Core Facility (CEMF) and Service for Single Particle (SSP) in 2019 and their Projects

1Daniela Nicastro, PhD

(Cell Biology)

CPRIT RR140082,

CPRIT RP170644,

NIGMS R01 GM083122 Mike Henne, PhD

(Cell Biology)

Michael Rosen, PhD

(Biophysics)

CPRIT (RR160082),

Welch F. I-1944-20180324

Xiaochen Bai, PhD

(Biophysics)2

Hongtao Yu, PhD

Project Titles and Cancer Relevance removed

11

Xiaochen Bai, Ph.D.

She J, Zeng W, Guo J, Chen Q, Bai XC*, Jiang Y* (2019) Structural

mechanisms of phospholipid activation of the human TPC12 channel. eLife

8:e45222 doi:10.7554/eLife.45222 (*corresponding authors).

Xiaochen Bai, Ph.D.

Chen Q, Zeng W, She J, Bai XC*, Jiang Y* (2019) Structural and functional

characterization of an otopetrin family proton channel. eLife 8:e46710 doi:

10.7554/eLife.46710 (*corresponding authors).

Xiaochen Bai, Ph.D.

Wang Y, Nguyen NX, She J, Zeng W, Yang Y, Bai XC*, Jiang Y* (2019)

Structural mechanism of EMRE-dependent gating of the human mitochondrial

calcium uniporter. Cell 177:1252-1261.e13. doi: 10.1016/j.cell.2019.03.050

(*corresponding authors).

4Xuewu Zhang, PhD

(Pharmacology)

NCI 1R01CA220283,

NIGMS R01 088197),

Welch foundation I-1702

experimentsZhijian Chen, PhD;

Xiaochen Bai, PhD1-3

1. Zhang C, Shang G, Gui X, Zhang X, Bai XC, Chen ZJ (2019) Structural basis

of STING binding with and phosphorylation by TBK1. Nature 567: 394-398.

2. Shang G, Zhang C, Chen ZJ, Bai XC, Zhang X (2019) Cryo-EM structures of

STING reveal its mechanism of activation by cyclic GMP-AMP. Nature 567: 389-

393.

Gharpure A, Teng J, Zhuang Y, Noviello CM, Walsh RM Jr, Cabuco R, Howard

RJ, Zaveri NT, Lindahl E, Hibbs RE (2019). Agonist Selectivity and Ion

Permeation in the α3β4 Ganglionic Nicotinic Receptor. Neuron doi:

10.1016/j.neuron.2019.07.030. [Epub ahead of print ] PMID: 31488329

4

Qi X, Liu H, Thompson B, McDonald J, Zhang C, Li X (2019) Cryo-EM structure

of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi. Nature

571:279-283.

1

7Jose Rizo-Rey, PhD

(Biophysics)

NINDS R35 NS097333,

Welch F. I-1304Daniela Nicastro, PhD

1. Quade B, Camacho M, Zhao X, Orlando M, Trimbuch T, Xu J, Li W, Nicastro

D, Rosenmund C, Rizo J (2019) Membrane bridging by Munc13-1 is crucial for

neurotransmitter release. eLife 8:e42806. [PMCID: PMC6407922]

2. Prinslow, E. A., Stepien, K. P., Pan, Y.-Z., Xu, J. and Rizo, J. (2019) Multiple

factors maintain assembled trans-SNARE complexes in the presence of NSF

and aSNAP. eLife 8, e38880 [PMCID: PMC6353594].

1 manuscript submitted

9Zbyszek Otwinowski, PhD

(Biophysics)

CPRIT RP180751,

HHS272201700060C,

R01GM117080,

R01GM118619,

P01AI120943,

DE-SC0019600,

R37HL072304

training, experiments

Bromberg R, Guo Y, Borek D, Otwinowski Z (2019) High-resolution cryo-EM

reconstructions in the presence of substantial aberrations. bioRxiv doi:

https://doi.org/10.1101/798280

10Hongtao Yu, PhD

(Pharmacology)

CPRIT RP150538-P2, Welch

Foundation (I-1441) training, experiments SBL 2,4 1 manuscript in preparation

11Jan Erzberger, PhD

(Biophysics)CPRIT RR150074 training, experiments 2 1 manuscript in preparation

12Weiwei Wang, PhD

(Biophysics)

Welch F. I-2020-20190330;

Endowed Scholars Program2 1 manuscript in preparation

1, 2

Helen Hobbs, PhD 2

2

Xiaochen Bai, PhD

6

14

Xiaochun Li, PhD

(Molecular Genetics)

Welch Foundation I-1957,

Endowed Scholars Program

UTSW, Damon Runyon-

Rachleff Innovation Award

(DRR-53-19), NIH grant

R01GM134700, NIH grant

R01GM135343

Ryan Hibbs, PhD

(Neurobiology)

NIDA R01DA042072,

NINDS R01NS095899,

NIDA R33DA037492

training

Bruce Beutler, MD

(Center for Genetics of

Host Defense)

NIAID U19AI00627,

NIAID R01AI125581training, experiments

13Daniel Rosenbaum, PhD

(Biophysics)

R01 GM113050,

R01 NS103939

Kim Orth, PhD

(Molecular Biology)8 HHMI

Youxing Jiang, Ph.D

(Pharmacology and

Biophysics)

HHMI, NIGMS GM079179,

Welch Foundation I-1578training3

5

training, experiments SBL

12

Hongtao Yu, PhD

Daniela Nicastro, PhD

16Xin Liu, PhD

(Obstetrics & Gynecology)

NIGMS GM121662,

NIGMS GM114576,

Welch Foundation I-1790

training, experiments 4

17Gui-Min LI, PhD (Radiation

Oncology)

NCI CA192003,

CPRIT RR160101,

NIGMS GM112702

experiments 2

18

Yunsun Nam, PhD (Green

Center for Reproductive

Biology Sciences)

NIGMS R01 GM122960 training 2

19Arun Radhakrishnan

(Molecular Genetics)

NHLBI P01 HL20948 (co-PI)

Welch Foundation I-1793Dan Rosenbaum, PhD 1, 2

20Xuelian Luo, PhD

(Pharmacology)

Welch I-1932;

R01-GM132275Xiaochen Bai, PhD 2

21

Lukasz Joachimiak, PhD

(Ctr for Alzhmrs Neuro

Disease)

endowed scholar training

22Ilya Bezprozvanny, PhD

(Physiology)

R01NS056224

R01AG055577 training Meewhi Kim, PhD,

23

Daniel Siegwart, PhD

(Simmons Compreh.

Cancer Center)

CPRIT RP190251,

ACS RSG‐17‐012‐01, NIBIB

RO1-EB025192, DoD

CA150245P3, Welch F.

I‐1855

full service

(experiments)SBL 1

24Elizabeth Chen, PhD

(Molecular Biology)NIAMS RO1-AR053173

full service

(experiments)SBL

25Yuh Min Chook, PhD

(Pharmacology)30+61:64

full service

(experiments)SBL 4

26Ian Corbin, PhD

(Internal Medicine)NCI RO1-CA214702

full service

(experiments)SBL 1,3

27Beth Levine, MD

(Internal Medicine)

CPRIT RP120718-P1,

NIAID U19-AI142784

full service

(experiments)SBL 1,2,3

28Deepak Nijhawan, MD/PhD

(Biochemistry)Peloton SRA

full service

(experiments)SBL 1,2

29Ruhma Syeda, PhD

(Neuroscience)

Endowed Scholar, American

Heart Association, Welch

Foundation

full service

(experiments)SBL 4

30Vincent Tagliabracci, PhD

(Molecular Biology)

CPRIT RR150033, NIGMS

DP2-GM137419, Endowed

Scholar, Welch Foundation I-

911, Searle Scholars

Foundation

full service

(experiments)SBL 1,2

Group 1: Structure-based drug design: Rational drug design based on protein structure is essential to the evolution of small molecules with high target affinity and minimal unwanted toxicities. Powered by an in-house high-throughput screening and medicinal chemistry facility that has for over a

decade identified small molecules targeting atypical anti-cancer intervention points, UTSW has recently seen several of these drug candidates enter clinical testing. In the future, use of the CEMF and SSP could optimize and expedite the cancer drug discovery pipeline at UTSW by providing high-

resolution cryo-EM structures of targets captured with newly discovered small molecules to guide modifications of the hit structure during the hit-to-lead and/or lead optimization phases.

Group 2: Cancer Relevant Drug Targets: Despite the well-established relevance of certain proteins to cancer, their value as therapeutic targets remains untested due to the absence of established drug-like small molecules that can modulate their activity. In the last decade, researchers at UTSW

have identified high priority anti-cancer targets using a multipronged approach, including mass spectrometry, high-throughput screening and metabolomics. High-resolution structures of potential targets provided by the CEMF and SSP will aid cancer researchers to determine protein architecture (e.g.

active/regulatory site, binding pockets) or uncover novel protein-protein interactions that support target protein activity in cancer-promoting cellular processes. These structural insights will strengthen the new anti-cancer drug development programs at UTSW that aim to disrupt target protein activity

either by direct chemical antagonism or elimination of cancer-promoting protein interactions.

Group 3: Immunotherapy: Decades of research efforts to harness the extraordinary power and exquisite specificity of the body’s natural immune system to attack and eliminate cancerous cells have recently seen milestone achievements for a number of cancers. The next crucial step in basic and

clinical research aims at extending anti-cancer immunotherapeutic responses to cancer types that have so far resisted. In the last year, UTSW has assembled an immunotherapy research group that consists of basic researchers and clinical scientists that oversee nearly 20 immunotherapy-related

ongoing clinical trials and that aim at advancing strategies for eliciting immune responses against cancerous cells. The CEMF will play a critical role in this research front by providing structural insights into drug targets, biologics and cellular pathways directly relevant to adaptive immunity and its

exploitation for anti-cancer treatments.

Group 4: Cellular mechanisms of tumor initiation and progression: The foundation of all robust cancer intervention strategies is the formulated attack of cell biological processes that have been hijacked in cancerous cells to promote unfettered growth. For decades, cancer cell biologists at

UTSW have led efforts to understand the cellular pathways and mechanisms that enforce coordinated cell growth in multicellular animals. With the support of the CEMF, investigators will use cryo-EM and in situ cryo-ET to visualize cellular structures, organelles and macromolecular complexes that

are co-opted in cancer cell survival. These efforts, when coupled with genetically-based interrogation of protein function, will reveal linchpin molecules in cancer-promoting cellular pathways and thus potential targets that can be exploited for novel anti-cancer treatments.

15Luke Rice, PhD

(Biophysics)

NIGMS R01 098543,

NSF MCB 1615938

13

14

4) Considerations concerning different data collection strategies (multi-hole/shot)

You might have seen the recent announcement by James Chen (SBL) that 9-hole multi-hole data acquisition is

now available on our Talos Arctica. Here are some relevant considerations to help CEMF users decide between

different data acquisition strategies.

4.1. Brief CEMF history concerning “multi-hole” (= images from multiple holes using Image Shift rather

than slower stage shift) and “multi-shot” (= multiple images from one hole):

2018 Krios: EPU used for multi-shot (mostly 3 images/hole) – [multi-hole not yet available in EPU]

Krios: switch to SerialEM because of faster multi-shot data acquisition

Talos: SerialEM – Dr. Otwinowski’s lab used distortion-UN-corrected 9-hole multi-hole data

2019/02 Krios: SerialEM 4-hole multi-hole with 3-image multi-shot setup (=12x) established

Talos: SerialEM 4-hole multi-hole (4x) available

Note: after visit to UCSF, decision was made against general 9-hole multi-shot strategy at the time,

because of worries about image distortions due to high image shift and unstable calibrations for

distortion-correction that required daily staff re-calibration at UCSF.

2019/03 Krios K3 install (~5x increased read-out speed)

2019/04 Krios: SerialEM 9-hole multi-shot setup (9x3 or 9x2) established on request of Dr. Xiaochun Li

(other users were trained to use this pattern upon request)

Talos: started ordering small C2 apertures (20 µm and 30 µm) to reduce beam width at parallel beam

illumination conditions to allow multi-shot (like on the Krios x12) [=> work in progress].

2019/10 Talos: SerialEM distortion-corrected 9-hole multi-shot setup (9x1) on request of James Chen (SBL)

2019/11 Talos: Conference call with FEI confirmed that PNCC (and UMass) use the same “30 µm C2 aperture

strategy” that we are pursuing to establish 2-image multi-shot on a Talos Arctica

Future: Krios: a new EPU version that is compatible with K3 camera will be installed this week and tested in

the coming weeks.

Talos: 30 µm C2 aperture will be tested for multi-shot; 20 µm C2 aperture (would allow 3-image multi-

hole) failed previously due to fringes, but as soon as Fringe-Free-Illumination becomes available

as purchasable upgrade for Talos will also revisit 20 µm C2 multi-shot.

4.2. Considerations about different data acquisition schemes:

Moving the beam to a different sample site for image acquisition using small amounts of Image Shift (IS) (also

called “beam-image shift”) is much faster and more accurate than using stage shift. Therefore, multi-shot (i.e. 2-4

images collected within one hole using small IS) and multi-hole (i.e. images collected from several neighboring

holes in a 4-hole, 5-hole or 9-hole pattern using moderate amounts of IS) can increase the speed of data

collection significantly (between 3x to 27x).

However, even in a well-aligned electron microscope, it is known that IS induces beam-tilt and with it a potential

structure phase error due to axial coma (= how much the incident direction of the electron beam is off from the

perfect optical axis, where a small angle results in the electron beam not hitting the specimen perpendicularly and

introducing phase errors).

Today’s image processing algorithms can correct for many aberrations introduced during the imaging

acquisition (see e.g. Bromberg R, Guo Y, Borek D, Otwinowski Z (2019) High-resolution cryo-EM

reconstructions in the presence of substantial aberrations. bioRxiv doi: https://doi.org/10.1101/798280).

However, depending on the origin of the introduced errors, some information might be difficult to recover or

un-recoverable by post-processing (especially when multiple aberrations from different sources are present),

and/or if not applied with caution, then corrections (=raw data alterations) making incorrect assumptions about

15

the origin of the aberrations could introduce more errors. [Note, if correcting large image shifts later by image

processing software (e.g. Relion) one should group the shifts into patches].

Therefore, an often applied strategy in cryoEM is to eliminate aberrations/errors experimentally (meaning

by hardware in the electron microscopes) as much as possible (“taking best possible” rather than “good

enough” data), and not leaving everything to post-processing corrections. Fortunately, data acquisition

softwares like SerialEM offer now more sophisticated and automated procedures to experimentally correct e.g.,

“coma vs IS” and more. However, one should keep in mind that experiences/studies about the data quality (e.g.

resolution of a center-hole reconstruction vs 9-hole-corner-hole reconstruction) and over-time stability of the

calibrations on a user-operated TEM (e.g. the calibrated Coma vs ImageShift Matrix) are very limited so far.

Therefore, users should consider the following when deciding on which data acquisition strategy to use: a) how

much worse do the data becomes relative to the amount of IS, b) how well is the coma corrected that is induced

by the image-beam shift (understand the process well enough to be able to judge quality of correction, because

current calibrations do not “fail” even if the found distortion correction solution is incorrect), c) “cost/benefit

evaluation” between data quality and speed of data collection in relation to target goals (e.g. are final data being

collected on the Krios or preliminary data on the Talos, and so on)?

In Cheng et al. 2018, the center hole reconstruction was compared to the corner reconstruction from a 5-hole

multi-hole image acquisition pattern on a 300kV Titan Krios: “a specimen with ultimate resolution of 2.75 Å

can tolerate 0.76 mrad of beam tilt (~ beam-image shift of +/- 3 to 4.5 µm) before its resolution is dropped

below 3 Å“ (Cheng A, Eng ET, Alink L, Rice WJ, Jordan KD, Kim LY, Potter CS, Carragher B. (2018) High

resolution single particle cryo-electron microscopy using beam-image shift. J Struct Biol. 204:270-275. doi:

10.1016/j.jsb.2018.07.015).

To my knowledge a similar comparison of center hole vs corner hole reconstructions has not been published for

a 9-hole pattern on a 200kV Talos (which will be worse than for a Krios, because the Talos has only two C2s

and limited alignment settings/correction possibilities). However, the Carragher lab generously shared the

following unpublished Glacios (same as Talos) data with us:

The slide (from Anchi Cheng)

shows residual beam tilt correction

needed (as detected by Relion 3.1)

for 5-hole data collected on a

Glacios, even after calibrated

“coma vs beam-image shift”

correction (note that the center

hole was “sacrificed” for focus

image on an Au-grid).

Anchi recommends that “for

maximal resolution, Relion 3.1

correction will do better than

hardware correction on a

Glacios/Talos, because the column

does not have the optical setup for

correcting higher order aberration

and the values can get high in

particular settings”.

In summary, know the pros and cons, test your data quality (i.e. center vs corner image reconstructions), and

choose the “best” data acquisition scheme based on your situation.

If you have questions about this topic and/or want to learn how to use the different acquisition schemes, please

ask the facility manager, Dr. Daniel Stoddard, or me.

With best regards, Dany Nicastro (CEMF Director)