Transformative High Resolution Cryo-Electron Microscopy Jon Lorsch, - - PowerPoint PPT Presentation

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Transformative High Resolution Cryo-Electron Microscopy Jon Lorsch, PhD Director National Institute of General Medical Sciences NIH Working Group: NEI, NIGMS, NCI, NHLBI, NIAID, NIDA, NIDDK, NINDS, ORIP, NIAAA, NIBIB Inter-agency Working

SLIDE 1

Transformative High Resolution Cryo-Electron Microscopy

Jon Lorsch, PhD Director National Institute of General Medical Sciences

NIH Working Group: NEI, NIGMS, NCI, NHLBI, NIAID, NIDA, NIDDK, NINDS, ORIP, NIAAA, NIBIB Inter-agency Working Group: NSF, DOE, Beckman

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Working Group Members

Co-Chairs Jon Lorsch (NIGMS) Cathy Lewis and Susan Gregurick Paul Sieving (NEI) Belinda Seto Working Group Coordinators: Mary Ann Wu (NIGMS) Jim Deatherage (NIGMS) Houmam Araj (NEI) OSC Ravi Basavappa Becky Miller Ellie Murcia IC Members Dan Gallahan (NCI) John (Randy) Knowlton (NCI) Alison Yao (NIAID) Rao Rapaka (NIDA) Salvatore Sechi (NIDDK) Paula Flicker (NIGMS) Denis Buxton (NHLBI) Manjit Hanspal (NHLBI) Jue Chen (NHLBI) James Luo (NHLBI) Margaret Sutherland (NINDS) Malgorzata Klosek (ORIP)

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Why Now? New Technological Breakthroughs in Cryo-EM

3 Old Methods 1) New electron microscopy technology dramatically improves our ability to see biological molecules New Methods

TRPV1 Ion Channel: Mediates burn sensation, Yifan Cheng UCSF

2) New motion correction methods resolve blurring of images due to movement of particles in electron beam

Rotavirus Particles Niko Grigorieff, Janelia Farms

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The U.S. is Rapidly Falling Behind Europe and Asia in Cryo-EM Infrastructure

Initial Investment, 1-2 CryoEM microscopes, local facility Significant Investment, 3-4 CryoEM microscopes, regional facility Major Investment, 5+ CryoEM microscopes, HTP comprehensive facility Planned investment

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# Request for Information (RFI): Transformative High-Resolution Cryo-Electron Microscopy

Notice Number: NOT-RM-16-022

Release Date: June 24, 2016 Response Date: August 8, 2016

Need and Capacity

Overwhelming support

for National Centers for automated high- resolution cryoEM data collection for SPA

US is falling behind
CryoET expertise,

resources also needed

Training and Workforce

Expertise in all stages

required for success

2 levels of trainees:

Structural biologists Neophyte biologists

Hands-on training at

Centers essential

Tutorials, online

materials at home Technology Development

HTP pipeline for thin

sections of cells

Optics and

instrumentation

Segmentation and

subtomogram averaging

Data management and

storage needs

, 46 responses

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Ebola virus neutralization by protective human antibodies (9Å)

Two human monoclonal antibodies, mAb100 and mAb114 in combination, protect nonhuman primates against all signs of Ebola virus disease.

Misasi et al. (2016) Science 351: 1343

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Zika Virus 3.8Å

7

Sirohi et al. (2016) Science 352: 467,

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HSP90-Cdc37-Cdk4 Complex at 3.9Å

8

HSP90 Interacts with

60% of human kinome.

Cdk4 kinase is trapped

and stabilized in the complex.

HSP90 inhibition leads

to degradation of kinases, including

ncoproteins vSrc,

bRafV600E, Her2.

HSP90 inhibitors are

undergoing clinical trials as cancer therapeutics.

Verba, K.A. et al. (2016) Science, 352:1542

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Challenges and Strategies

Challenges ● Cost of instrumentation and upkeep

Access to high performance data collection ●

Limited base of expert investigators Strategy ● Open access to state of the art instrumentation ● Build an expert workforce ● Improve and extend technology ● Create economies of scale Comprehensive Centers ● Three centers ● Research assistance and training 20-30 labs/year ● High-throughput data collection services ● Each center 4 microscopes, 7-8 FTEs, $4M TC/year ● R01/R21s to develop new technology & methods Long Term Plan ● High throughput data collection services at three centers, each $2M TC/year ● Interagency WG (NIH, NSF, DOE, Beckman)

Glutamate dehydrogenase (1.8A) Merk et al. Cell 165, 1698, 2016

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Proposed Budget

10

Comprehensive Centers (3)

Year 1 Year 2 Year 3 Year 4 Year 5

Total Equipment (4 microscopes/center)

$22M $22M $22M

$66M Operations

$4M $6.4M $10.5M $10.5M $10.5M

$41.9M User Training & Service

(4 FTEs/center)

$0.6M $1.2M $2.5M $2.5M $2.5M

$9.3M

$26.6M $29.6M $35M $13M $13M

$117.2M

Research Grants (R21,R01)

TC Yearly

Total Cryoelectron Tomography R&D

$3M

$15M

Single Particle R&D

$2.4M

$12M

$5.4M

$27M All Activities