Protein crystallography in practice
MCB
15 Dec 2016
Daved H. Fremont [email protected]
Department of Pathology and Immunology Washington University School of Medicine
An 7-step program for protein structure determination by x-ray crystallography
1. Produce monodisperse protein either alone or as relevant complexes 2. Grow and characterize crystals 3. Collect X-ray diffraction data 4. Solve the phase problem either experimentally or computationally 5. Build and refine an atomic model using the electron density map 6. Validation: How do you know if a crystal structure is right? 7. Develop structure-based hypothesis
1. Produce monodisperse protein either alone or as relevant complexes
Methods to determine protein purity, heterogeneity, and monodispersity! Gel electrophoresis (native, isoelectric focusing, and SDS-PAGE)! Size exclusion chromatography! Dynamic light scattering http://www.protein-solutions.com/
! Circular Dichroism Spectroscopy http://www-structure.llnl.gov/cd/cdtutorial.htm
Characterize your protein using a number of biophysical methodsEstablish the binding stoichiometry of interacting partners �
2. Grow and characterize crystals
! Hanging Drop vapor diffusion ! Sitting drop, dialysis, or under oil ! Macro-seeding or micro-seeding ! Sparse matrix screening methods
! Random thinking processes, talisman, and luck The optimum conditions for crystal nucleation are not
necessarily the optimum for diffraction-quality crystal growth
Space Group P214 M3 /ASU
diffraction >2.3Å14.4% Peg6K
NaCacodylate pH 7.0200mM CaCl2
Space Group P31213 M3 + 3 MCP-1/ASU
diffraction > 2.3Å18% Peg4K
NaAcetate pH 4.1100mM MgCl2
Space Group C22 M3 /ASU
diffraction >2.1Å18% Peg4K
Malic Acid/Imidazole pH 5.1
100mM CaCl2
Commercial screening kits available from http://www.hamptonresearch.com; http://www.emeraldbiostructures.com
Hanging Drop Sitting drop
No Xtals? ���
Decrease protein heterogeneity
! Remove purification tags and other artifacts of protein production
! Remove carbohydrate residues or consensus sites (i.e., N-x-S/T)
! Determine domain boundaries by limited proteolysis followed by mass spectrometry or amino-terminal sequencing. Make new expression constructs if necessary.�
! Think about the biochemistry of the system! Does your protein have co-factors, accessory proteins, or interacting partners to prepare as complexes? Is their an inhibitor available? Are kinases or phosphatases available that will allow for the preparation of a homogeneous sample?�
! Get a better talisman
Building a crystal
a
b
c
α
β
γ
a
b
c
αβ γ
The unit cell
Crystal symmetries
A triclinic lattice (no symmetry)
Crystal symmetries
Introducing a twofold axis produces a monoclinic lattice P2
Crystal symmetries
The threefold axis generates a trigonal crystal - but now α=β=90o, γ=120o
and a = b
Crystal symmetries
We cannot fill space with a fivefold arrangement – although the asymmetric unit can contain
a fivefold axis (e.g. virus capsids)
These restrictions give rise to 7 crystal classes in 3 dimensions
The seven crystal classes
3. Collect X-ray diffraction data ! Initiate experiments using home-source x-ray generator and detector
! Determine liquid nitrogen cryo-protection conditions to reduce crystal decay ! While home x-rays are sufficient for some questions, synchrotron radiation is preferred ! Anywhere from one to hundreds of crystals and diffraction experiments may be required
Argonne National Laboratory Structural Biology Center beamlineID19 at the Advanced Photon Source http://www.sbc.anl.gov
3. Collect X-ray diffraction data Lawrence Berkeley National LaboratoryALS Beamline 4.2.2
4. Solve the phase problem either experimentally or computationally
! Structure factor equation:
! By Fourier transform we can obtain the electron density. We know the structure factor amplitudes after successful data collection. Unfortunately, conventional x-ray diffraction doesn’t allow for direct phase measurement. This is know as the crystallographic phase problem.
! Luckily, there are a few tricks that can be used to obtain estimates of the phase α(h,k,l)
Experimental Phasing Methods ! MIR - multiple isomorphous replacement - need heavy atom incorporation
! MAD - multiple anomalous dispersion- typically done with SeMet replacement ! MIRAS - multiple isomorphous replacement with anomalous signal ! SIRAS - single isomorphous replacement with anomalous signal
Computational Methods
! MR - molecular replacement - need related structure ! Direct and Ab Initio methods - not yet useful for most protein crystals
Diffraction DataP1
SeMet-1P1
SeMet-2P1
SeMet-3P1
SeMet-4P21
Native
Wavelength (Å) 1.07813 0.97956 0.97945 0.94645 0.97945
Resolution (Å) 100-1.60 100-1.60 100-1.60 100-1.60 100-1.40
Number of sites 4 4 4 4 ---
Reflections measured (unique)
126 734(49 265)
146 273(56 587)
146 405(56 619)
149 455(57 749)
495 848(45 711)
Completeness overall (outer shell)
81.8(35.3)
93.9(80.4)
94.0(80.9)
95.8(94.8)
96.1(82.1)
I/σ (I) overall (outer shell)
22.5(7.9)
26.8(11.6)
21.9(9.8)
19.1(7.3)
23.0(3.0)
Rsym(%) overall (outer shell)
5.9(19.2)
5.8(17.0)
6.0(19.3)
6.5(28.6)
10.0(67.2)
Rcullis (20-1.6 Å) iso/ano ---/0.967 0.359/0.563 0.283/0.529 0.607/0.729 ---Phasing power iso/ano ---/0.818 4.56/3.10 6.19/3.30 0.819/2.31 ---
Resolution Number of
reflections/ Number ofprotein atoms/ Rcrystal/ Rms Deviations
Refinement Range (Å) completion solvent atoms Rfree (%) Bonds (Å) Angles (o)
P1-SeMet 20.0-1.60 (1.66-1.60)
29 053/ 97.1%(2750/95.7%))
1957/266 16.8/21.1(26.2/27.8)
0.010 1.9
P21-native 20.0-1.40 (1.45-1.40)
45 632/ 96.3%(3965/84.6%))
1957/244 17.8/20.9(26.0/26.3)
0.011 1.5
MAD phasing statistics for the AP-2 α-appendage
Electron density for the AP-2 α-appendage
Initial bones trace for the AP-2 α-appendage
Final trace for the AP-2 α-appendage
5. Build an atomic model using the electron density map
What does a good map look like?
Before computers, maps were contoured on stacked pieces of plexiglass. A “Richards box” was used to build the model.
half-silvered mirror
plexiglass stack
brass parts model
Low-resolution
At 4-6Å resolution, alpha helices look like sausages.
Medium resolution
~3Å data is good enough to see the backbone with space in between.
Holes in rings are a good thing
Seeing a hole in a tyrosine or phenylalanine ring is universally accepted as proof of good phases. You need at least 2Å data.
The resolution of the electron-density map and the amount of detail that can
be seen Resolution Structural Features Observed 5.0 Å Overall shape of the molecule 3.5 Å Ca trace 3.0 Å Side chains 2.8 Å Carbonyl oxygens (bulges) 2.5 Å Side chain well resolved,
Peptide bond plane resolved 1.5 Å Holes in Phe, Tyr rings 0.8 Å Current limit for best protein
crystals
The 2.8 Å density of SrfTE
The 2.8 Å density of SrfTE could be skeletonised
and traced
6. Validation: How do you know if a crystal structure is right?
The R-factor R = Σ(|Fo-Fc|)/Σ(Fo)
where Fo is the observed structure factor amplitude and Fc is calculated using the atomic model. R-free
An unbiased, cross-validation of the R-factor. The R-free value is calculated with typically 5-10% of the observed reflections which are set aside from atomic refinement calculations.
Main-chain torsions: the Ramachandran plot
Geometric Distortions in bond lengths and angles
Favorable van der Waals packing interactions
Chemical environment of individual amino acids
Location of insertion and deletion positions in related sequences
6. Validation: How do you know if a crystal structure is right?
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of theα-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.Natl. Acad. Sci. U.S.A. 96:8907-8912.
6. Validation: Mapping of sequence conservation in AP-2 α-subunit appendages
Structure-Based Mutagenesis of the α-appendage
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of theα-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.Natl. Acad. Sci. U.S.A. 96:8907-8912.
7. Develop structure-based hypothesis
Example: West Nile Virus
About 70 members, half of which are associatedwith human disease (Yellow fever, Japanese encephalitis)
Enveloped, spherical virion, 40 - 50 nm in size
Three structural proteins: C,M (prM) and E ; seven non-structural proteins (NS1-5)
ssRNA genome, linear, positive polarity, 11 kb,infectious
C M E NS1 NS2a 2b NS3 NS4a 4b NS5
Structural proteins Non-structural proteins
5’UTR 3’UTR
Production of soluble E proteins and ectodomain fragments
Immunize mice with soluble E (25 µg x 3)
Fuse splenocytes with myeloma line
Large panels of flavivirus mAbs
Table 1. Summary of Data Collection and RefinementData Collection for West Nile Virus Envelopea
Space Group P41212Unit Cell (Å3) a=89.6 b=89.6 c=154.0Wavelength(Å) 0.90X-ray Source APS-BM 14Resolution(Å) (outer shell) 20-2.9 (3.08-2.90)Observations/Unique 14408/62790Completeness(%) 98.5 (99.5)Rsym(%) 5.7 (52.4)I/σ 16.9 (2.05)Refinement Statisticsb
Resolution(Å) (outer shell) 20-3.0 (3.19-3.00)Reflections Rwork/Rfree 11506/607#Protein Atoms/Solvent/Heterogen 3031/28/38Rwork overall(outer shell) (%) 26.2(35.6)Rfree overall(outer shell) (%) 30.8(34.1)Rmsd Bond lengths (Å)/angles(o) 0.008/1.6Rmsd Dihedral/Improper (o) 24.9/0.84Ramachandran plotMost Favored/Additional (%) 78.2/21.8Generous/Disallowed (%) 0.0/0.0Average B-values 92.0Est. Coordinate Error (Å) 0.47 aValues as defined in SCALEPACK (Otwinowski and Minor, 1997).
bValues as defined in CNS (Brunger, AT)
Structure Determination of WNV Envelope Protein
Envelope Protein and the Flavivirus virion
DIII
DI
DII
X-ray crystal structure of E
Immature
5
3 2prM Cleavage
Mature
60 trimers of prM/E heterodimers 180 E monomers
Cryo-EM model of WNV
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
3 3.5 4 4.5 5 5.5
PFU
/g o
f tis
sue
Day 9 - E16 Day 9 - PBS
0 5 10 15 20 25 300
20
40
60
80
100
E16 (2 mg)
E24 (2 mg)
PBS
Treatment Day 5
Days Post Infection
E16 is a potent neutralizing mAb with therapeutic activity against WNV in mice
Single Dose mAb at Day 5 Post-Infection
Humanized E16 binds WNV DIII with similar affinities and kinetics as E16
-10
-5
0
5
10
15
20
25
30
35
40
45
-50 0 50 100 150 200 250 300 350 400
RU
Response
sTime
DIII binding E16
-10
-5
0
5
10
15
20
25
30
-50 0 50 100 150 200 250 300 350 400Time s
Response
RU
DIII binding Hm-E16.3
Antibody ka (1/Ms) kD (1/s) Rmax KD (nM) Chi2
E16 1.1 x 106 0.0118 39.5 10.8 0.33Hm-E16.1 9.6 x 105 0.0201 32.8 21.0 0.16Hm-E16.2 1.0 x 106 0.0092 24.7 9.2 0.13Hm-E16.3 9.9 x 105 0.0070 24.1 7.1 0.16
Summary of Surface Plasmon Resonance (SPR) studies
Bacterial expression of WNV E Domain 3
E16 Fab by papain cleavage
mAb capture by Protein A
Elution Volume (ml)
Abs 2
80 (m
AU)
DIII-E16 Fabcomplex
DIII alone
Complex purification by size exclusion chromatography
Production and purification of DIII in complex with E16 Fab
Hybridoma expression of E16 mAb
Refolding of DIII
DIII
Structure determination of DIII-E16 complex by X-ray crystallography
Data collection for D3-E16 complex
Space Group P212121 Unit Cell (Å3) a=52.4 b=83.3 c=110.6 X-ray Source ALS Resolution(Å) (outer shell) 30-2.50 (2.59-2.50) Observations/Unique 59923/16985 Completeness(%) 97.6 (82.7) Rsym(%) 8.3 (30.6) I/ 11.3 (2.7) Atomic refinement statistics Rwork overall(outer shell) (%) 20.8(25.6) Rfree overall(outer shell) (%) 28.2(31.8) Ramachandran plot Most Favored/Additional (%) 87.5/11.9 Generous/Disallowed (%) 0.4/0.2
CH
VH
CL
VL
Structure of the DIII-E16 Fab complex
Nybakken et al, Nature 2005
DIII
DIIIE16 Fab
N-terminal region
BC Loop
DE Loop
FG Loop
L2
L1L3
H3
H2
H1
E16 Fab
VH VL
Selection of E16 specific epitope variants of DIII
Yeast library of DIII variantscreated by error prone PCR
DIII mutations at Ser306, Lys307, ThrE330 and Thr332 significantly diminish E16 binding
Pooled
DIII
mAbs
E16 staining
E -DIII
DIII yeast display mutations are centrally located at the E16 interface
DIII
ThrE332
ThrE330
LysE307
SerE306
CH
VH
CL
VL
DIII
E16 Fab
LysE307
SerE306
TrpH33
SerH95
H1 H3
ThrE332
ThrE330LysE307H2 ArgH58
AspH100
DIII N-Term
H3
DIII BC loop
DIII N-Term
WNVE DIII
E16 Fab C
C
NN
N
C
BCDEFG WNVE
DII
E53 Fab CL
CH1
VH VL
C
C
NN
1A1D-2 Fab
DV2E DIII N
C
BCDE FG
CL
CH1
VH
CL
CH1
VHVL
C
C
NN
Ser 306
Lys 306Thr 330
Thr 332
Leu 107
Gly 106
Arg 99
Pro 75Thr 76
Lys 305
Lys 307
Lys 310
Fusion loop
AB
Yeast display ≤ 4.5 Å contacts
VL
E16 Fab could potentially bind 120/180 E protein DIII sites on WNV
Zhang, et al, Nat Structural Biology, 2003Mukhopadhyay, et al, Science, 2003
Fusion Loop
E16 binding to 2- and 3-fold clustered DIIIs appears permissivewhile 5-fold clustered DIII binding appears sterically non-permissive
Combination of crystallographic and cryo-EM data - the E16 Fab/WNV complex
Cryo-EM reconstruction ofE16 Fab complex with WNV
Model of E16 Fab complex with WNV
Cryo-EM work done in collaboration with Rossmann and Kuhn groups at Purdue University
Fitting E16 Fab complex into Cryo-EM reconstruction of WNV
Cross-section of Cryo-EM reconstruction
E16 internalizes with the virus during infection of vero cells
E53 E16
DIC / Bright Field
FluorescentMerge
Alexa 488-labeled WNV mAbs and lysotracker red (acidified endosomes)
Pre bind virus + Alexa-Ab
Add to cells at 4 or 37oC
15 minutes. Fix, addLyso-trackerConfocal microscopy
E16 Fab decoration appears to trap WNV particles - a fusion intermediate?
pH 6pH 8
nucleocapsid core (~ 154Å) n ucleocapsid core (~ 158Å) outer lipid layer (~200 Å ) o uter lipid layer (~205Å) outer glycoprotein layer (~245Å) outer density layer (~340Å)
www.sciencemag.org SCIENCE VOL 314 22 DECEMBER 2006 1875
Aquaculture in Offshore Zones
THE EDITORIAL BY ROSAMOND NAYLOR,“Offshore aquaculture legislation” (8 Sept.,p. 1363), suggests that the motivation formoving aquaculture into the open ocean isthat “marine f ish farming near the shoreis limited by state regulations.” Althoughunworkable regulations may exist in a fewstates, in the larger scheme this is irrele-vant. Of the offshore aquaculture projectscurrently under way, none are occurring inthe U.S. Exclusive Economic Zone (EEZ);rather, they are happening in state waters.Even historically, only two aquacultureprojects have ever occurred in federalwaters (1).
Much of Naylor’s stated concern overoffshore aquaculture is based on historicalexperience with near-shore fish farms. Thisis in spite of years of more relevant offshore
operations that reveal little, if any, negativeimpact on the environment or local ecosys-tems (2, 3). Naylor criticizes the NationalOffshore Aquaculture Act of 2005 becauseit lacks specific environmental standards.Yet, she recommends California’s recentSustainable Oceans Act as a legislativemodel, although it is similarly silent, leavingthose details to rule-making in response tothe best available science.
Naylor criticizes the use of fishmeal asan aquaculture ingredient, ignoring the factthat industrial fisheries are well managedand would occur with or without aquacul-ture’s demand. Naylor ignores the higherefficiency of using fishmeal to feed fishcompared with its use in land-based live-stock operations (4). Also ignored is theinefficiency of using small pelagic fish inthe natural setting to feed predator fish (5).
Researchers and entrepreneurs currentlydeveloping the technologies needed for offshoreaquaculture share a vision of a well-managed
industry governed by regulations with a rationalbasis in the ecology of the oceans and the eco-nomic realities of the marketplace.
CLIFFORD A. GOUDEY
Massachusetts Institute of Technology, Cambridge, MA02139, USA.
References and Notes1. The SeaStead project a decade ago, four miles off
Massachusetts (see www.nmfs.noaa.gov/mb/sk/saltonstallken/enhancement.htm) and the recentOffshore Aquaculture Consortium experimental cageoperation 22 miles off Mississippi (see www.masgc.org/oac/).
2. See www.lib.noaa.gov/docaqua/reports_noaaresearch/hooarrprept.htm/.
3. See www.blackpearlsinc.com/PDF/hoarpi.pdf.4. See www.salmonoftheamericas.com/env_food.html.5. D. Pauly, V. Christensen, Nature 374, 255 (2002).
IN HER PROVOCATIVE EDITORIAL “OFFSHOREaquaculture legislation” (8 Sept., p. 1363),R. Naylor raises valid points regarding regu-lation of oceanic aquaculture, since it issure to grow in the future because of dwin-dling global fishery supplies. This growth is
LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES
1878
Generating new sciencein the classroom
How proteins connect
1880 1882
Mathematicalperspectives
LETTERSedited by Etta Kavanagh
Retraction
WE WISH TO RETRACT OUR RESEARCH ARTICLE “STRUCTURE OFMsbA from E. coli: A homolog of the multidrug resistance ATP bind-ing cassette (ABC) transporters” and both of our Reports “Structure ofthe ABC transporter MsbA in complex with ADP•vanadate andlipopolysaccharide” and “X-ray structure of the EmrE multidrug trans-porter in complex with a substrate” (1–3).
The recently reported structure of Sav1866 (4) indicated that ourMsbA structures (1, 2, 5) were incorrect in both the hand of the struc-ture and the topology. Thus, our biological interpretations based onthese inverted models for MsbA are invalid.
An in-house data reduction program introduced a change in sign foranomalous differences. This program, which was not part of a conven-tional data processing package, converted the anomalous pairs (I+ andI�) to (F� and F+), thereby introducing a sign change. As the diffrac-tion data collected for each set of MsbA crystals and for the EmrEcrystals were processed with the same program, the structures reportedin (1–3, 5, 6) had the wrong hand.
The error in the topology of the original MsbA structure was a con-sequence of the low resolution of the data as well as breaks in the elec-
tron density for the connecting loop regions. Unfortunately, the use ofthe multicopy refinement procedure still allowed us to obtain reason-able refinement values for the wrong structures.
The Protein Data Bank (PDB) files 1JSQ, 1PF4, and 1Z2R forMsbA and 1S7B and 2F2M for EmrE have been moved to the archiveof obsolete PDB entries. The MsbA and EmrE structures will berecalculated from the original data using the proper sign for the anom-alous differences, and the new C⇥ coordinates and structure factorswill be deposited.
We very sincerely regret the confusion that these papers havecaused and, in particular, subsequent research efforts that were unpro-ductive as a result of our original findings.
GEOFFREY CHANG, CHRISTOPHER B. ROTH,
CHRISTOPHER L. REYES, OWEN PORNILLOS,
YEN-JU CHEN, ANDY P. CHEN
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
References1. G. Chang, C. B. Roth, Science 293, 1793 (2001).2. C. L. Reyes, G. Chang, Science 308, 1028 (2005).3. O. Pornillos, Y.-J. Chen, A. P. Chen, G. Chang, Science 310, 1950 (2005).4. R. J. Dawson, K. P. Locher, Nature 443, 180 (2006).5. G. Chang, J. Mol. Biol. 330, 419 (2003).6. C. Ma, G. Chang, Proc. Natl. Acad. Sci. U.S.A. 101, 2852 (2004).
COMMENTARY
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Chang G, Roth CB. (2001) Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293(5536):1793-800. PMID 11546864Pornillos O, Chen YJ, Chen AP, Chang G. (2005) X-ray structure of the EmrE multidrug transporter in complex with a substrate. Science 310(5756):1950-3. PMID 16373573Reyes CL, Chang G. (2005) Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide. Science 308(5724):1028-31. PMID 15890884Chang G. (2003). Structure of MsbA from Vibrio cholera: a multidrug resistance ABC transporter homolog in a closed conformation. J Mol Biol 330(2):419-30. PMID 12823979Ma C, Chang G. (2004). Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli. Proc Natl Acad Sci USA 101(9):2852-7. PMID 14970332
When things go wrong:
Structure of the Amino-Terminal Protein Interaction Domain of STAT-4Uwe Vinkemeier, Ismail Moarefi, * James E. Darnell Jr., John KuriyanScience 13 February 1998:Vol. 279. no. 5353, pp. 1048 - 1052
Figure 2. Tertiary structure of the N-domain of STAT-4. (A) Overall representation of two monomers (green and gray) in the crystallographic dimer, viewed approximately orthogonal to the molecular twofold axis, which is vertical. The ring-shaped NH2-terminal element is colored red in one monomer. (B) Orthogonal view of one of the N-domains shown in (A), depicting details of the architecture of the ring-shaped element. Side chains that participate in a charge-stabilized hydrogen-bond network are shown in a ball-and-stick representation. The side chain and backbone carbonyl of buried R31 are shown in magenta. For clarity, the indole ring of the invariant residue W4 that seals off this arrangement on the proximal side is drawn with thinner bonds. The blue sphere denotes a buried water molecule. Hydrogen bonds are indicated by dotted lines. Oxygen, nitrogen, and carbon atoms are red, blue, and yellow, respectively. Q3-N marks the position of the backbone amide group of residue Q3. The light-red segment of helix 2 highlights its 310 helical conformation. Fig. 2 and Fig. 3, B and C were created with the program RIBBONS, version 2.0 (28).
Figure 1. Analysis of STAT4 dimers produced by crystallographic symmetry to identify the physiologic dimer.(a) Dimer A (produced by the fractional transformation -Y, -X, -Z+1/6 with translation 1, 1, 1) represents the dimer implied previously22. Dimer B (produced by the fractional transformation X, X−, -Z+5/6 with translation 0, 1, 0) represents an alternative interface recently suggested25. Highlighted residues were targeted for mutational studies. Residues W37, T40, and E66 (magenta) are located in the dimer A interface, whereas residues D19 and L78 (cyan) are located in the dimer B interface. (b) Surface analysis of the two dimers. According to this analysis, dimer B is a statistically better molecular interface (as compared to dimer A) and is more likely to represent a physiologically relevant dimer.
Naruhisa Ota, Tom J Brett, Theresa L Murphy, Daved H Fremont & Kenneth M Murphy N-domain-dependent nonphosphorylated STAT4 dimers required for cytokine-driven activation Nature Immunology 5, 208 - 215 (2004)