link to slides

Opportunity and Challenge:
the new era of cryo-EM
Ning Gao
School of Life Sciences
Tsinghua University
2015-10-20
Education and Research Experience
Principal Investigator
2008.11-present
Postdoc, Department of Biochemistry and
Biophysics, and HHMI (Advisor: Joachim Frank)
2006-2008
Ph.D., Biomedical Sciences
2001-2006 (Advisor: Joachim Frank)
B.S. Biochemistry and Molecular Biology
1996-2000
Research Interest
• Ribosome biogenesis
– In vivo assembly pathways and quality control of ribosome
maturation
– Molecular roles of biogenesis factors
• Translation regulation
• Ribosomes from human pathogens
– Understanding Pathogen-specific translation mechanisms
– Screening for small molecules with preventive and
therapeutic potentials.
• Large RNA/DNA-protein complexes
– DNA replication initiation
• Mechano-sensitive channels
Representative Structural Snapshots
Ribosome
MCM2-7
Piezo1
Near atomic structure of a ribosomal
complex
Resolution
4.5 Å
From 300,000
particles,
CCD data
Nature Methods 11, 30 (2014)
CCD vs EDD
CCD
Electron Direct
Detector
From Gatan website
Counting mode and Super-resolution
From Gatan website
Movie Mode and Motion Correction
Bronze Age  Iron Age
• Better detective quantum
efficiency (signal-to-noise ratio)
• Faster speed
• High Sensitivity
Kühlbrandt, 2014, Science
Unpublished data
Small membrane channel
Unpublished data
800 micrographs and 180,000 particles
in 17 hours
70S ribosome (3.0 Å)
Unpublished data
Opportunities
Challenges
YnaI : a small-conductance
mechanosensitive channel (MscS)
Yoo et al., Biophys, 2009
Sense mechanical deformation to regulate osmotic pressure.
A heptamer with ~ 250 kDa in size.
Negative staining of YnaI
Aggregate in 1 hr
Preferred orientation
Elusive Side-views
1.2/1.3 grid
no carbon coated
350 micrographs (K2 camera)
≈130,000 particles
>93% top views
1500 micrographs 600,000 particles
Atomic model of YnaI (3.6 Å)
a
Trans-membrane
region
Cytoplasmic
region
b
Atomic model + Density map
c
Atomic Model
Unpublished data
Sample preparation (traditional)
3-5 l
Liquid
ethane
-172°C
Liquid N2
-196°C
Ethane: - 88.6 (boiling point), -172 (melting
point), cooling speed very fast
Traditional EM grids
Holey carbon
grid
• Standardized cryo-sample preparation
– Smaller sample volume
– Better particle distribution
– Reproducibility and manipulability
Microfluidics + fast screening by LM
Piezo: mechano-transduction in
eukaryotes
Coste et al., 2010, Science
Putative topology of Piezo1
Nilius, 2010, EMBO Reports
Negative Staining of mPiezo1
2D class averages
Cryo-EM of mPiezo1
3Å
FFT
100 nm
Mapping the extracellular CAP domain
Nilius, 2010, EMBO Reports
3D structure of Piezo1 channel
CAP
Top
Bottom
Side
Sub-region Refinement
Fitting of crystal structure of the CAP
Flexible Refinement
Original Particles
Modified Particles
TM-helices resolved by FlexRef
Original Map
Map from
modified particles
Transmembrane Wings
Pore Module of mPiezo1
PH PH
PH PH PH1
Anchor
OH
CED
IH
CTD
Gating Mechanism of mPiezo1
CED
Blade
Out
PH
In
OH IH
Anchor
CTD
Beam
Ge et al., 2015, Nature, accepted
A major challenge:
heterogeneous data sorting
• Defined number of conformations
• Highly dynamic system
– Multiple (independent) moving parts
Divide, conquer, and stitch
Projection Theorem
z2
p( x, y )   o( x, y, z )dz o(x,y,z) : 3D density distribution of the object
z1
From Frank, 2002. Annu. Rev. Biophys. Biomol. Struct.
31:303-19
Mathematical Description of
molecular flexibility
• Multiple conformers of a molecular structure can be
expressed by modulation of the original density map
by deformation functions, D1, D2, D3…Dn, which
normally can be expressed by a rotation matrix and a
pair of translational parameters (x, y) describing the
conformational changes of individual rigid (relatively)
domains.
• During refinement for each particle , in addition to
orientation parameters (x, y,  ,  ,  ), parameters of
Dn could also be estimated.
Acknowledgments
Ministry of Science and Technology, China
National Natural Sciences Foundation of China
Ministry of Education, China
Collaborators:
THU:
Jianlin Lei
Maojun Yang
Bailong Xiao
Jijie Chai
Bik Tye (HKUST & Cornell)
Suparna Sanyal (Uppsala)
John Woolford (CMU)
Hyouta Himeno (Hirosaki)
Yan Qin (IBP)
EM Facility Setup and Maintainance:
Senfang Sui, Hongwei Wang, Xueming Li
Jianlin Lei, Yanji Xu
Thank you！
Structural Snapshots of Actively
Translating Human Ribosomes
Behrmann et al., 2015, Cell
Behrmann et al., 2015, Cell